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 181 records · Page 10Linked to original sources
PubMed2005

Induction of mitochondrial fusion by cysteine-alkylators ethacrynic acid and N-ethylmaleimide.

Mitochondrial fusion and fission are important aspects of eukaryotic cell function that permit the adoption of varied mitochondrial morphologies depending upon cellular physiology. We previously observed that ethacrynic acid (EA) induced mitochondrial fusion in cultured BSC-1 and CHO/wt cells. However, the mechanism responsible for it was not clear since EA has a number of known cellular effects including glutathione (GSH) depletion and alkylation of cysteine residues. To gain insight, we have tested the effects of a variety of compounds on EA induced cellular toxicity and mitochondrial fusion. N-acetyl cysteine (NAC), a GSH precursor, was found to abrogate both the toxic and fusion-inductive effects, whereas diethylmaleate (dEM), a GSH depletor, potentiated both these effects in a dose-dependent manner. However, treatment with dEM alone, which depleted GSH to the same degree as EA, did not induce mitochondrial fusion. These results indicate that although detoxification of EA via formation of GSH conjugates is dependant upon GSH levels, the depletion of GSH by EA is not responsible for its effect on mitochondrial fusion. Dihydro-EA (DH-EA), a saturated EA analogue, lacked EA's toxicity and effect on fusion, indicating that the alpha,beta-unsaturated ketone is central to its observed effects. N-ethylmaleimide (NEM), another well-known cysteine-alkylator, also induced mitochondrial fusion at near toxic concentrations. These data suggests that cysteine-alkylation is the causative factor for fusion and toxicity. In live BSC-1 cells, EA induced fusion of mitochondria occurred very rapidly (<20 min), which suggests that it is inducing fusion by modifying certain critical cysteine residue(s) in proteins involved in the process.

Animals↗
PubMed1980

Regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide.

The regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide (NEM) was investigated using the agonist ligand, [3H] cis methyldioxolane ([3H]CD). Characterization studies on rat forebrain homogenates show that [3H]CD binding was linear with tissue concentration and was unaffected by a change in pH from 5.5 to 8.0. The regional variation in [3H]CD binding in the rat brain correlated generally wit [3H](-)3-quinuclidinyl benzilate ([3H](-)QNB) binding, although the absolute variation in binding was somewhat less. At a concentration of 100 microM, the GTP analogue, guanyl-5'-yl imidodiphosphate [Gpp(NH)p], caused a 43-77% inhibition of [3H]CD binding in the corpus striatum, ileum, and heart. The results of binding studies using several Gpp(NH)p concentrations demonstrated that the potency of this guanine nucleotide for inhibition of [3H]CD binding, Gpp(NH)p caused an increase in (3H](-)QNB binding in the heart heart and ileum and no change in [3H](-)QNB binding in the corpus striatum. When measured by competitive inhibition of [3H](-)QNB binding to the longitudinal muscle of the ileum, Gpp(NH)p (100 microM) caused an increase in the IC50 values of a series of agonists in a manner that was correlated with the efficacy of these compounds. The results of binding studies on NEM treated forebrain homogenates revealed an enhancement of [3H]CD binding by NEM.

Animals↗
PubMed2004

The effect of N-ethylmaleimide on transmitter release from the skeletal neuromuscular junction of Bufo marinus.

N-ethylmaleimide (NEM) has been used extensively in biochemical assays as an inhibitor of the NEM sensitive fusion protein (NSF). However, examination of the effect of NEM on transmitter release in more physiologically relevant preparations has proved inconclusive. In the present study, we have examined the effect of low concentrations of NEM on synaptic transmission in intact nerve-muscle preparations from toads (Bufo marinus). Under conditions of low transmitter release probability (0.3 mM calcium, 1 mM magnesium), treatment with NEM (10 microM) caused a significant increase in the amplitude of stimulus-evoked endplate potentials (EPPs) and a significant increase in the frequency of spontaneously occurring miniature EPPS (MEPPS) without affecting the amplitude of MEPPs. When the calcium concentration in the bath was raised to 4 mM, 10 microM NEM had no effect on EPP amplitude. Under these conditions, NEM treatment reduced paired pulse facilitation and increased depression during stimulus trains. Treatment with NEM also resulted in a significant decrease in the synaptic delay. The effects of NEM on transmitter release in the present study were not due to inactivation of G-proteins. The results of the present study show a calcium-dependent facilitation of stimulus-evoked transmitter release by NEM. These results are discussed in terms of the possible sites of NEM action leading to the observed changes in transmitter release.

Animals↗
PubMed1995

N-ethylmaleimide profiling of yeast NADP-dependent isocitrate dehydrogenase.

Yeast NADP-dependent isocitrate dehydrogenase is inactivated by N-ethyl-maleimide (NEM) at pH 7.7 and 30 degrees C. Reaction with cysteine382 occurs most rapidly and is accompanied by loss of about 50% of the enzymatic activity. A slower phase of inactivation ensues during which lysine343 is the major target of NEM, while minor products result from reaction at cysteine73 and cysteine354. Protection against the second phase of inactivation is provided by NADP, NAPH, or manganous-isocitrate. Comparison of the time-dependence of inactivation and the products of reaction with N-ethyl-ethylmaleimide (NEM profiling) of the pig heart (G. E. Smyth and R. F. Colman, 1991, J. Biol. Chem. 266, 14918-14925) and yeast NADP-specific isocitrate dehydrogenases have been coupled with an examination of the crystal structure of the Escherichia coli isocitrate dehydrogenase. The following conclusions have been reached: while no cysteine is essential for activity, yeast Cys382/pig Cys379 is close to the adenine portion of the NADP binding site, and pig Cys269 is located in the region of the metal-isocitrate binding site.

Amino Acid Sequence↗
PubMed1995

Phosphorylation of wheat germ initiation factor 2 (eIF-2) by N-ethylmaleimide-treated wheat germ lysates and by purified casein kinase II does not affect the guanine nucleotide exchange on eIF-2.

Phosphorylation of the small subunit of eukaryotic initiation factor-2 (eIF-2 alpha) impairs protein synthesis in mammalian systems. It is not known, however, if a similar regulatory mechanism exists in plants. Previous reports indicate that one of the wheat germ eIF-2 subunits, the p40-41 doublet, is phosphorylated by heterologous eIF-2 alpha kinases. Here we report that phosphorylation of the small subunit in wheat germ eIF-2, p36, occurs in translating wheat germ lysates which are pretreated with N-ethylmaleimide (NEM) and dithiothreitol. Also, a purified sea star casein kinase II (CKII) phosphorylates the p41-42 doublet and p36 subunits of wheat germ eIF-2. While heme-regulated eIF-2 alpha kinase from reticulocyte lysates does not inhibit wheat germ protein synthesis, CKII and NEM are found to be inhibitory. To determine whether phosphorylation of the small subunit (p36) is the cause for protein synthesis inhibition, we have further studied the exchange of labeled GDP for unlabeled GDP in the preformed eIF-2. [3H]GDP complex in vitro in the presence of CKII and ATP. The GDP exchange in eIF-2.GDP complex can occur without the addition of any protein factor and the exchange reaction is marginally inhibited by CKII. A 0-70% ammonium sulfate cut fraction, prepared from NEM-treated wheat germ lysate, also does not inhibit the guanine nucleotide exchange reaction. These findings suggest that the protein synthesis inhibition in these cases is not mediated by eIF-2 phosphorylation.

Casein Kinase II↗
PubMed1999

N-Ethylmaleimide-modified Hsp70 inhibits protein folding.

Hsp70 molecular chaperones facilitate protein folding and translocation by binding to hydrophobic regions of nascent or unfolded proteins, thereby preventing their aggregation. N-Ethylmaleimide (NEM) inhibits the ATPase and protein translocation-stimulating activities of the yeast Hsp70 Ssa1p by modifying its three cysteine residues, which are located in its ATPase domain. NEM alters the conformation of Ssa1p and disrupts the coupling between its nucleotide- and polypeptide-binding domains. Ssa1p and the yeast DnaJ homolog Ydj1p constitute a protein folding machinery of the yeast cytosol. Using firefly luciferase as a model protein to study chaperone-dependent protein refolding, we have found that NEM also inhibits the protein folding activity of Ssa1p. Interestingly, the NEM-modified protein (NEM-Ssa1p) is a potent inhibitor of protein folding. NEM-Ssa1p can prevent the aggregation of luciferase and stimulate the ATPase activity of Ssa1p suggesting that it acts as an inhibitor by binding to nonnative forms of luciferase and by competing with them for the polypeptide binding site of Ssa1p. NEM-Ssa1p inhibits Ssa1p/Ydj1p-dependent protein refolding at different stages indicating that the chaperones bind and release nonnative forms of luciferase multiple times before folding is completed.

Adenosine Triphosphatases↗
PubMed2000

Modification of cysteine residues by N-ethylmaleimide inhibits annexin II tetramer mediated liposome aggregation.

A role of cysteine residues in annexin II tetramer (AIIt)'s function was investigated using the sulfhydryl reagent N-ethylmaleimide (NEM). Incubation of AIIt with NEM resulted in a dose-dependent inhibition of AIIt-mediated liposome aggregation and loss of sulfhydryl groups of AIIt. The concentration effecting 50% inhibition was 0.18 mM. The inhibition was observed in all Ca2+ concentrations tested (1-1000 microM). NEM had no effects on liposome aggregation mediated by other annexins (I, III, and IV), indicating that the inhibitory effect caused by NEM modification is specific to AIIt. The NEM-treated AIIt still can bind to liposomes. However, once AIIt was bound to membrane, the cysteine residues were protected from NEM modification. Our results suggest that cysteine residues are critical for AIIt-mediated liposome aggregation.

Animals↗
PubMed2002

Translation and phosphorylation of wheat germ lysate: phosphorylation of wheat germ initiation factor 2 by casein kinase II and in N-ethylmaleimide-treated lysates.

Previously, we observed that N-ethylmaleimide (NEM), a thiol-alkylating agent, was found to stimulate the phosphorylation of several proteins in translating wheat germ (WG) lysates, including the phosphorylation of alpha, the p41-42 doublet subunit, and beta, the p36 subunit, of the WG initiation factor 2 (eIF2). We find now that NEM increases phosphorylation of several proteins significantly in lysates which are moderate or low in their translation compared to optimally active lysates. Heat treatment, which stimulates oxidation of protein sulfhydryls, decreases the translation and phosphorylation ability of WG lysates. The decrease in phosphorylation, but not translation, that occurs in heat-treated lysates is prevented very efficiently by NEM and partially by reducing agents such as dithiothreitol (DTT) and GSH. DTT prevents, however, completely the loss of sulfhydryl content of heat-treated WG lysates and does not at all prevent heat-induced inhibition of translation. In contrast, DTT prevents completely the diamide-induced translational inhibition and also the loss of sulfhydryl content. These findings therefore suggest that in addition to the maintenance of sulfhydryl groups, heat-labile proteins and their interactions with other proteins play an important role in overall translation and phosphorylation. It is also observed here that heat treatment stimulates the phosphorylation of rabbit reticulocyte eIF2 alpha but not the alpha subunit (p41-42 doublet) of WG eIF2. A phosphospecific anti-eIF2 alpha antibody recognizes the WG eIF2 alpha(P) that is phosphorylated by an authentic eIF2 alpha kinase such as double-stranded RNA-dependent protein kinase, but it is unable to recognize the eIF2 alpha that is phosphorylated in NEM-treated lysates. These findings therefore suggest that phosphorylation of WG eIF2 alpha in NEM-treated lysates occurs on a site different from the serine 51 residue that is phosphorylated by authentic eIF2 alpha kinases. In addition, it also suggests that WG eIF2 alpha, unlike reticulocyte eIF2 alpha, is phosphorylated by eIF2 alpha kinases and also by other kinases. Consistent with this idea, it has been observed here that casein kinase II (CKII) phosphorylates WG eIF2 alpha and the phosphorylation is enhanced by NEM in vitro and in lysates. The phosphopeptide analysis suggests that WG eIF2 alpha has separate phosphorylation sites for CKII and heme-regulated eIF2 alpha kinase (a well-characterized mammalian eIF2 alpha kinase), and NEM-induced phosphorylation in WG lysates resembles CKII-mediated phosphorylation.

Casein Kinase II↗
PubMed2000

N-ethylmaleimide-sensitive factor is associated with the nuclear envelope.

N-Ethylmaleimide-sensitive factor (NSF) is an ATPase involved in many membrane fusion events within the exocytic and endocytotic pathways. In the present study we showed that NSF is associated with the nuclear envelope. Golgi-associated NSF was released from membranes upon incubation with Mg(2+)-ATP, reflecting the disassembly of a complex consisting of NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). In contrast nuclear envelope-associated NSF in interphase cells was not released by the same treatment. During mitosis, however, it was released from nuclear membranes by Mg(2+)-ATP. These results suggest that the binding mode of nuclear membrane-associated NSF changes during the cell cycle.

Adenosine Triphosphate↗
PubMedDate not supplied

Pertussis toxin and N-ethylmaleimide inhibit histamine- but not calcium ionophore-induced endothelium-dependent relaxation.

Endothelium-dependent relaxation of the guinea pig pulmonary artery induced by histamine was inhibited by preincubation of the tissue with 10 microM N-ethylmaleimide (NEM) for 10 min, whereas the endothelium-dependent relaxation induced by the calcium ionophore A 23187 was not affected by NEM. Pretreatment of the preparations with 0.2-1 microgram/ml pertussis toxin for 120 min inhibited concentration-dependently the histamine-induced relaxation. In contrast, endothelium-dependent relaxation in response to the calcium ionophore A 23187 was not affected by pertussis toxin. Since NEM and pertussis toxin are thought to interfere with membrane located GTP binding proteins, it is suggested that such a coupling protein is involved in the signal transduction of the histamine receptor leading to endothelium-dependent relaxation.

Animals↗
PubMed1988

Inhibition of neuronal noradrenaline uptake (uptake1) and desipramine binding by N-ethylmaleimide (NEM).

The inhibition of N-ethylmaleimide (NEM) of uptake1 and desipramine binding was studied on clonal rat phaeochromocytoma cells (PC12 cells) in different experimental settings: (1) 3H-noradrenaline uptake into intact PC12 cells; (2) 3H-noradrenaline uptake into isolated PC12 plasma membrane vesicles; (3) 3H-desipramine binding to isolated PC12 plasma membrane vesicles. In plasma membrane vesicles, NEM inhibited 3H-desipramine binding and 3H-noradrenaline uptake with similar potency (the IC50's were 1.36 mmol/l and 1.04 mmol/l, respectively). However, in intact cells, NEM was about 75 times more potent in inhibiting 3H-noradrenaline uptake (IC50 = 0.014 mmol/l). The increased potency of NEM in intact cells is probably due to an inhibition of the Na+/K+-ATPase and not to a direct interaction with the noradrenaline carrier. The inactivation by NEM of 3H-desipramine binding to PC12 plasma membrane vesicles was irreversible. Both an inhibitor (cocaine, 1 mmol/l) and a substrate of uptake1 (amezinium, 1 mmol/l) protected desipramine binding from inactivation. These results are compatible with the hypothesis of a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier.

Adrenal Gland Neoplasms↗
PubMed1991

Inhibition of the muscarinic receptor-activated K+ current by N-ethylmaleimide in rabbit heart.

The effects of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, on the ACh-activated K+ current were examined in single cells from rabbit hearts using whole-cell and single channel patch clamp techniques. Bath application of NEM (50 microM) or the muscarinic antagonist, atropine (1 microM) completely inhibited the ACh-activated K+ current in whole-cell recordings. In cell-attached patch conditions, the inhibitory effect of NEM was still observed; in contrast, atropine was ineffective when the agents were bath applied, indicating that the site of action of NEM is different from that of atropine. Inside-out patch recordings confirmed that GTP was required on the internal side of the membrane for activation of this K+ channel when ACh was present in the pipette. NEM abolished this GTP-activated K+ channel activity. GTP gamma S, a non-hydrolysable GTP analogue, was able to activate this K+ channel in the absence of a muscarinic agonist, an effect thought to be due to the direct activation of GTP-binding proteins. Pretreatment with NEM almost completely prevented this effect of GTP gamma S. In contrast, after the activation of the K+ channel by GTP gamma S had reached a steady-state, NEM failed to show a significant inhibitory effect. These results demonstrate that NEM prevents the activation of muscarinic receptor-regulated K+ channel and suggest an involvement of alkylation of the GTP-binding proteins which are coupled to this type of K+ channel.

Acetylcholine↗
PubMed1992

Incorporation of 3H-N-ethylmaleimide into sheep red cell membrane thiol groups following protection by diamide-induced oxidation.

The thiol oxidant diazene dicarboxylic acid bis [N,N-dimethylamide] (diamide) is known to reversibly activate K-Cl cotransport in sheep red blood cells. Although the detailed mechanism of activation is unknown, functional thiols at the membrane or at the cytoplasmic level are recognized as important. To search for membrane bound thiols involved in the regulation of K-Cl cotransport, sheep red cells were first exposed to diamide at concentrations activating K-Cl cotransport, and then to the alkylating agent N-ethylmaleimide (NEM) in order to block non-oxidized thiols. White ghosts, prepared by osmotic lysis from these cells, were again treated with NEM followed by reduction of the diamide-induced dithiols with dithiothreitol (DTT) concentrations known to reverse the diamide-induced K-Cl flux. Maximum 3H-NEM incorporation into the DTT-reduced thiols occurred at 50 microM DTT. Saturation labelling by 3H-NEM of about 2 x 10(4) diamide-protected thiols/cell occurred at 25 microM NEM. Diamide protected about 0.1% of all membrane thiols chemically determined earlier. Membranes from high K (HK) and low K (LK) sheep red cells did not differ significantly in the number of diamide-protected thiols, and polyacrylamide gels revealed a similar protein distribution of 3H-NEM-labelled thiols. Since diamide is known to stimulate K-Cl flux in LK cells ten times more than in HK cells this finding is consistent with the hypothesis of a cytoplasmic control effecting different K-Cl flux activities in the membranes of the two cation genotypic red blood cells.

Animals↗
PubMed1984

Effects of N-ethylmaleimide on adenosine receptors of rat fat cells and human platelets.

N-Ethylmaleimide (NEM) differentially modified Ri adenosine receptors in rat fat cells and Ra adenosine receptors in human platelets. Pretreatment of rat fat cell membranes with NEM inhibited the binding of the agonist (-)N6-phenylisopropyl[3H]adenosine [( 3H]PIA), but did not affect the binding of the antagonist 1,3-diethyl-8-[3H]phenylxanthine [( 3H]DPX). The IC50-value for inhibition of [3H]PIA binding was 0.067 mM. Saturation of [3H]PIA binding revealed that NEM converts the high affinity form of the Ri receptor into a low affinity form. NEM also decreased the potency of agonists to displace [3H]DPX binding, as shown by a 74-fold shift of the Ki-value for (-)PIA, whereas antagonist-induced displacement remained unchanged. In addition, low concentrations of NEM (0.01-0.1 mM) attenuated the (-)PIA-induced inhibition of adenylate cyclase activity of rat fat cells. At higher concentrations (0.1-1 mM) NEM reduced basal and stimulated adenylate cyclase activities in rat fat cells and human platelets, presumably by inactivation of the catalytic unit. Radioligand binding of 5'-N-ethylcarboxamido[3H]-adenosine [( 3H]NECA) to Ra adenosine receptors of human platelet membranes was not changed by NEM at low radioligand concentrations. Saturation analysis of [3H]-NECA binding showed that NEM led to an apparent increase of agonist affinity with a concomitant decrease in total [3H]NECA binding sites. These results suggest that NEM reduces the affinity of Ri adenosine receptors, probably by affecting the inhibitory guanine nucleotide binding protein (Ni), whereas [3H]NECA binding sites are inversely affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1986

N-ethylmaleimide (NEM) diminishes alpha 2-adrenoceptor mediated effects on noradrenaline release.

The effect of N-ethylmaleimide (NEM), which has been shown to abolish rather selectively inhibition of adenylate cyclase, on the alpha 2-adrenoceptor-mediated modulation of noradrenaline release was studied. Slices of the rabbit hippocampus were loaded with 3H-noradrenaline, superfused continuously and stimulated twice electrically. NEM (30 mumol/l) applied for 30 min enhanced both basal and stimulation-evoked tritium overflow significantly. Occupation of the receptor by the alpha 2-adrenoceptor agonist clonidine prior to and during NEM treatment did not protect the alpha 2-adrenoceptor-mediated autoinhibitory feedback system from being affected by NEM. Preincubation of the hippocampal slices with NEM was without any influence on 3H-noradrenaline uptake. The inhibitory effect of clonidine on 3H-noradrenaline release was attenuated in a non-competitive manner. In addition, the facilitatory effect of the alpha 2-adrenoceptor antagonist yohimbine on the stimulus-evoked tritium overflow was reduced. The facilitation of the evoked noradrenaline release by yohimbine or yohimbine or yohimbine and NEM converged with increasing concentrations of yohimbine, suggesting that yohimbine and NEM were acting at the same signal-transduction system. These results are compatible with the idea that NEM, by alkylating the Ni-unit of a presynaptically located adenylate cyclase, prevents the alpha 2-adrenoceptor-mediated modulation of noradrenaline release.

Animals↗
PubMed1986

Discrimination by N-ethylmaleimide between the chronotropic and inotropic response to muscarinic receptor stimulation in rat atrium.

N-ethylmaleimide (NEM) rapidly blocked the negative chronotropic effect of carbachol on rat right atrium. In contrast, NEM did not reduce the negative inotropic response to muscarinic (M) receptor stimulation. Carbachol inhibited the specific binding of [3H]-N-methylscopolamine [( 3H]-NMS) to membranes of rat atria as reflected by a shallow inhibition curve. Both guanosine triphosphate (GTP) and NEM shifted the [3H]-NMS inhibition curves of carbachol to the right. Pretreatment of the atrial membranes with NEM abolished the GTP-induced rightward shift. However, when instead of the membranes the intact atria were pre-incubated with NEM, no interaction between NEM and GTP in the membranal preparation was observed. The results indicate that NEM sharply discriminated between the inotropic and chronotropic effects to M-receptor stimulation in rat atria. The inhibitory effect of NEM on the M-receptor-mediated negative chronotropic effect in rat atrium cannot be explained by an interaction of the sulfhydryl reagent with GTP-binding proteins, like Ni or No.

Animals↗
PubMed1988

Dual effect of N-ethylmaleimide on Cl- transport across the thin ascending limb of Henle's loop.

Effects of SH reagents on Cl- transport were studied in the isolated hamster thin ascending limb of Henle's loop (TAL) perfused in vitro. Parachloromercuribenzene sulfonate (PCMBS) at 10(-4) M in the bath decreased the relative permeability for Cl-/Na+ (PCl/PNa), as determined by the transmural diffusion voltage (VT) generated under a NaCl concentration gradient, from 2.71 +/- 0.16 to 1.11 +/- 0.09 (P less than 0.001). The effect of PCMBS was prevented by the pretreatment with 10(-3) M dithiothreitol (DTT). N-Ethylmaleimide (NEM) at 10(-3) M in the bath exhibited a dual action on Cl- permeability of the TAL: It inhibited the Cl- permeability in fresh preparations, whereas it stimulated the Cl- permeability in the preparations pretreated with SH reagents including NEM, maleimide and PCMBS. The inhibitory effect was irreversible but the stimulatory effect was reversible. Both responses were prevented by DTT. Since dextran-maleimide did not show any inhibitory effect on PCl/PNa, the SH site responsible for the inhibition may be located inside of the cell. The stimulatory effect of NEM on PCl/PNa was markedly reduced when bath pH was reduced to 5.8. On the other hand, when the bathing fluid was made nominally Ca2+ free, the stimulatory effect of NEM was unaffected, although the basal level of PCl/PNa was reduced These observations suggest that the conductive Cl- pathway in the TAL is either stimulated or inhibited by modifying two distinct SH sites. The site of modulation by proton binding may exist distally to these SH sites. The regulatory mechanism involving Ca2+ may be independent of the SH regulatory sites.

4-Chloromercuribenzenesulfonate↗
PubMed1988

Effects of the sulfhydryl reagent N-ethylmaleimide on reserpine binding to the catecholamine transporter in chromaffin granule membranes.

1. Catecholamines are transported into chromaffin granules via a carrier-mediated, active-transport process which is inhibited by micromolar concentrations of the sulfhydryl reagent, N-ethylmaleimide (NEM). Reserpine is a very potent, competitive inhibitor of the catecholamine transporter and can be used to investigate the characteristics of the catecholamine transporter. 2. The purpose of this study was to determine whether [3H]reserpine binding to the catecholamine transporter present in chromaffin granule membranes isolated from bovine adrenal glands was also inhibited by NEM and, if so, whether this was a direct or an indirect effect of NEM on the catecholamine transporter. 3. Both [3H]norepinephrine transport into and [3H]reserpine binding to the chromaffin granule ghosts isolated from bovine adrenal glands are inhibited by NEM, with IC50 values of 0.63 +/- 0.02 and 2.8 +/- 0.66 microM, respectively. 4. Mg and ATP protected both the [3H]norepinephrine transport into the ghosts and the [3H]reserpine binding to the transporter from inhibition by NEM, shifting the IC50 values to 260 +/- 43 and 120 +/- 29 microM, respectively. 5. NEM inhibition of the catecholamine transport and reserpine binding appears to be due to an action on the proton translocator associated with the Mg ATPase enzyme rather than a direct action on the catecholamine transporter since (a) the concentration of NEM required to inhibit formation of a membrane potential is similar to that required to inhibit [3H]norepinephrine transport into and [3H]reserpine binding to the ghosts and (b) Mg and ATP protected the proton translocation and [3H]norepinephrine transport into the ghosts, and [3H]reserpine binding to the ghosts, from inhibition by NEM.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗