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At least 109 records · Page 6Linked to original sources
PubMed2001

CD28 stimulation regulates its association with N-ethylmaleimide-sensitive fusion protein and other proteins involved in vesicle sorting.

CD28 delivers a co-stimulatory signal for T cell antigen receptor induced activation of T cells through a mechanism which remains mostly elusive to date. In order to try and gain insight into CD28 function, we therefore applied state-of-the-art mass spectrometric protein identification technology to the analysis of CD28 immunoprecipitates prepared from Jurkat T cells. We found that N-ethylmaleimide-sensitive fusion protein (NSF) and other proteins with sequence similarities to proteins part of or implicated in vesicular protein sorting pathways, were associated with CD28 in a CD28 stimulation-dependent manner. Furthermore, N-ethylmaleimide treatment abolished the NSF/CD28 interaction completely, and blocked CD28 association with a tyrosine phosphorylated 103 kDa protein in the activated cells. These results are suggestive of a potential model for CD28 co-stimulation regulated by an NSF-catalyzed mechanism.

Amino Acid Sequence↗
PubMed1993

Evidence for a common binding site for omeprazole and N-ethylmaleimide in subunit A of chromaffin granule vacuolar-type H(+)-ATPase.

Vacuolar-type H(+)-ATPase from adrenal chromaffin granules was found to be sensitive to omeprazole, a known gastric H+/K(+)-ATPase inhibitor, the concentration required for 50% inhibition being 80 microM freshly-prepared and 12 microM acid-treated reagent. ATP and ADP protected the enzyme from inhibition by omeprazole. The activity of the inhibited enzyme was restored by the addition of reduced glutathione. Omeprazole protected the enzyme from inhibition by N-ethylmaleimide and its binding to the subunit A. As subunit A has a nucleotide binding site(s) and as a cysteine residue is involved in the inhibition by N-ethylmaleimide, these results suggested that the two sulfhydryl reagents bind to the same cysteine residue near the nucleotide binding domain in the subunit A, resulting in inactivation of vacuolar-type H(+)-ATPase.

Adenosine Diphosphate↗
PubMed1980

Phosphate carrier of liver mitochondria: the reaction of its SH groups with mersalyl, 5,5'-dithio-bis-nitrobenzoate, and N-ethylmaleimide and the modulation of reactivity by the energy state of the mitochondria.

The inhibitory effect of three SH reagents, mersalyl, 5,5'-dithio-bis-nitrobenzoate, and N-ethylmaleimide, on Pi transport in rat liver mitochondria was investigated under a variety of conditions. Mersalyl binds at room temperature with both high (Kd less than 10 microM) and low affinity to mitochondria. Inhibition of Pi transport by mersalyl goes in parallel with titration of the high-affinity sites, inhibition being complete when 3.5-4.5 nmol/mg protein is bound to the mitochondria. At concentrations of mersalyl equal to or higher than 10 microM, inhibition of Pi transport occurs in less than 10 sec. At concentrations of mersalyl lower than 10 microM, the rate of reaction with the Pi carrier is considerably decreased. At a concentration of 100 microM, 5,5'-dithio-bis-nitrobenzoate fully inhibits Pi transport in about 1 min at room temperature. Nearly total inhibition is attained when as little as 40-50 pmol/mg is bound to mitochondria. Upon incubation longer than 1 min, additional SH groups, not belonging to the Pi carrier, begin to react. The uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone decreases the rate of reaction of mersalyl, 5,5'-dithio-bis-nitrobenzoate, and N-ethylmaleimide with the Pi carrier. Preincubation with Pi has a similar effect. We propose that both carbonyl cyanide p-trifluoromethoxyphenylhydrazone and Pi act by increasing the acidity of the mitochondrial matrix. Protonation of the Pi carrier at the matrix side would change the accessibility of its SH groups at the outer surface of the inner membrane. This might correspond to a membrane-Bohr effect, possibly related to the opening of a gating pore in the Pi carrier.

Animals↗
PubMed1983

Thiol-dependent passive K/Cl transport in sheep red cells: II. Loss of Cl- and N-ethylmaleimide sensitivity in maturing high K+ cells.

A fraction of the passive, ouabain-insensitive K+ fluxes in mature low Kr(LK) but not in high K+ (HK) sheep red cells requires the presence of Cl- anions and can be stimulated by volume expansion (Dunham, P.B., Ellory, J.C., J. Physiol (London) 318:511-530, 1981) or treatment with 2 mM N-ethylmaleimide (NEM) (Lauf, P.K., Theg., B.E., Biochem. Biophys. Res. Commun. 92:1422-1428, 1980). In the present study it is shown that reticulocytes of both anemic LK and HK sheep possess the Cl- -dependent K+ transport system which subsequently remains functional in mature LK but not in HK red cells. Kinetically, Cl- -mediated K+ fluxes of reticulocytes of LK sheep are different from mature red cells only in their Vmax values as measured in Na+ or choline+ media, while there is an apparently much lower affinity for external K+ ions in reticulocytes of HK sheep. N-ethylmaleimide stimulated K+ transport in the reticulocytes of both sheep genotypes suspended in Cl- but failed to do so in NO3- media. The data are interpreted in terms of their biochemical, physiologic, and genetic implications for the HK/LK transition in sheep red cells.

Anemia↗
PubMed1981

Evidence for a two-state mobile carrier mechanism in erythrocyte choline transport: effects of substrate analogs on inactivation of the carrier by N-ethylmaleimide.

Choline transport in erythrocytes is irreversibly inhibited by N-ethylmaleimide. The hypothesis that the carrier alternates between outward-facing and inward-facing forms and that only the latter reacts with the inhibitor (Martin, K. (1971) J. Physiol. (London) 213:647--667; Edwards, P.A. (1973) Biochim. Biophys. Acta 311:123--140) is here subjected to a quantitative test. In this test the effects of a series of substrate analogs upon rates of inactivation and rates of choline exit are compared. By hypothesis the effect of an analog in the external solution on the inactivation rate depends only on how it affects the proportion of the inward-facing carrier. Since 14C-choline efflux is necessarily proportional to the concentration of free carrier in the inward-facing form, the analogs should have related effects on the two rates. In every case the observed effects were identical, whether the analogs accelerated transport or inhibited it. Analysis of the results demonstrates that (1) the transport mechanism depends on the operation of a mobile element; (2) distinguishable inward-facing and outward-facing conformations of the free carrier, carrier-substrate complex, and carrier-inhibitor complex exist, and only the inward-facing forms react at a significant rate with N-ethylmaleimide; (3) carrier mechanisms involving a single form of free carrier or a single form of carrier-substrate complex are ruled out; and (4) dissociation of the carrier-substrate complex is a rapid step with all substrate analogs.

Biological Transport, Active↗
PubMed1981

Reaction of internal forms of the choline carrier of erythrocytes with N-ethylmaleimide: evidence for a carrier conformational change on complex formation.

The choline carrier of human erythrocyte membranes exists in distinguishable outward-facing and inward-facing conformations, and previous studies demonstrated that only the latter reacts with N-ethylmaleimide, producing an irreversible inhibition of transport. We now report experiments to determine the individual reaction rates for the two inward-facing forms: the free carrier and the complex. The pseudo-first-order rate constant for the complex with a substrate analog, di-n-butylaminoethanol, is found to be nearly double that for the free carrier, showing that the carrier conformation is altered following addition of a ligand (with 1 mM N-ethylmaleimide at pH 6.8, 37 degree C, the constants are 0.57 +/- 0.05 min-1 and 0.33 +/- 0.02 min-1, respectively). Hence three different conformational states have been distinguished by experiment: (1) the inward-facing free carrier; (2) the inward-facing complex; and (3) the outward-facing carrier.

Choline↗
PubMed1985

Chemical modification of the Na+/H+ exchanger of thymic lymphocytes. Inhibition by N-ethylmaleimide.

A Na+/H+ exchanger is involved in the regulation of cytoplasmic pH and cellular volume in a variety of cells. Little is known about the molecular nature of this exchanger. The purpose of this study was to survey a variety of group-specific covalent reagents as potential inhibitors of the exchanger. Na+/H+ countertransport activity was assayed as the amiloride-sensitive rate of Na+-induced alkalinization in acid-loaded lymphocytes, or as the rate of swelling in cells suspended in sodium propionate medium. Activity was not affected by proteinases or by carboxyl-group and amino-group specific reagents. A significant inhibition was produced by diethylpyrocarbonate, a histidine-specific reagent and by N-ethylmaleimide, a sulfhydryl group reagent. A similarly reactive but nonpermeating sulfhydryl agent, glutathione-maleimide, failed to inhibit Na+-H+ exchange. Moreover, the reaction with N-ethylmaleimide was sensitive to changes in the cytoplasmic pH. The data suggest that the chemically reactive groups of the Na+/H+ exchanger of lymphocytes have limited exposure to the extracellular medium but that an internally located sulfhydryl group is critical for the cation-exchange activity.

Animals↗
PubMed1980

Inactivation of Escherichia coli acetate kinase by N-ethylmaleimide. Protection by substrates and products.

Acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) from Escherichia coli exhibited a time-dependent loss of activity when incubated with N-ethylmaleimide at micromolar concentrations. However, prolonged incubation did not eliminate all catalytic activity and generally about 15% of its initial activity remained. When incubated with 7.2 microM N-ethylmaleimide, acetate kinase was inactivated with a rate constant of 0.063 min-1. Adenine nucleotides, ATP, ADP and AMP, protected the enzyme against such inactivation, but acetate up to 3.0 M and in the presence of 0.2 M MgCl2 and acetyl phosphate at 24 mM did not interfere with the rate of inactivation. While both acetate and acetyl phosphate did not affect the protection rendered by AMP, the presence of acetyl phosphate altered ADP protection. However, both substrates prevented ATP from protecting the enzyme. These data suggest that the binding sites for acetate and acetyl phosphate are different from that of the adenosine binding domain, but are in close vicinity to the phosphoryl binding regions of the nucleotides.

Acetate Kinase↗
PubMed1985

N-ethylmaleimide causes mechanical fragility and accumulation of spectrin dimers in the rat erythrocyte membrane.

Treatment of rat erythrocytes with N-ethylmaleimide is found to render them mechanically fragile. Membranes of the lysed cells show degradation of band 3 and, to a lesser extent, of spectrin; as well as considerable accumulation of dimeric spectrin. The predominant action of N-ethylmaleimide on isolated membranes, however, is the conversion of spectrin to its dimeric form.

Animals↗
PubMed1992

Inhibition of the light-induced H+ release from uncoupled thylakoid membranes by N-ethylmaleimide.

The light-induced H+ release from thylakoids, which can be observed under completely uncoupled conditions, was inhibited by the SH reagent N-ethylmaleimide (NEM) and its analogs, while the conventional H+ uptake and electron transfer were not affected. The half-inhibiting concentration of NEM for the H+ release was 10 mM and 4 mM in thylakoids in the presence of nigericin and in CF1-depleted thylakoids, respectively. The inhibitory effect increased with the increase in hydrophobicity of the NEM analogs: N-methylmaleimide less than N-ethylmaleimide less than N-phenylmaleimide. It is suggested that SH groups in hydrophobic interior within the membrane are essential to the release of protons.

Chloroplasts↗
PubMed1994

Modulation of dopamine D3 receptor binding by N-ethylmaleimide and neurotensin.

GTP or G protein inactivation by N-ethylmaleimide reduced the Bmax value but not the KD value of 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin ([3H]7-OH-DPAT) binding in the rat subcortical limbic area. Neurotensin (10 nM) increased the KD and the Bmax values of [3H]7-OH-DPAT binding, and these effects persisted also following N-ethylmaleimide pretreatment. N-Propylnorapomorphine, quinpirole, raclopride, and remoxipride inhibited [3H]7-OH-DPAT binding with Ki values of 0.093, 1.97, 10.6, and 710 nM, respectively. These findings indicate that the D3 receptor is coupled to G proteins in the brain, and that neurotensin can modulate D3 agonist binding by a G protein-independent mechanism.

Animals↗
PubMed1995

Interactions of N-ethylmaleimide and aluminium fluoride with GABAB receptor function in rat neocortical slices.

Interactions of N-ethylmaleimide and aluminium fluoride (AlF - 4) with GABAB receptors have been examined using spontaneously discharging rat neocortical slices. The suppression of discharges by the GABAB receptor agonist baclofen (5-10 mu M) was irreversibly prevented by N-ethylmaleimide (10-50 mu M) and its analog N-phenylmaleimide (10-50 mu M), whilst superfusion of slices with NaF (10 mM) and AlCl3 (100 mu M) to form a fluoroaluminate (AlF - 4) complex markedly potentiated the action of baclofen. The lipoxygenase inhibitors, nordihydroguaiaretic acid (10-50 mu M) and eicosatetraynoic acid (10-50 mu M) or the phospholipase A2 inhibitor bromophenacylbromide (50-100 mu M) did not affect the response to baclofen. The depressant action of baclofen is evidently mediated through G-proteins, but is not dependent on arachidonic acid metabolites.

Aluminum Compounds↗
PubMed1986

Effect of N-ethylmaleimide on rat liver phosphatidylcholine-specific and non-specific transfer protein activities: its dependence on donor liposome composition.

Phosphatidylcholine exchange between liposomes and mitochondria catalyzed by rat liver phosphatidylcholine transfer protein is strongly stimulated by N-ethylmaleimide (NEM) when PC/PI (molar ratio, 4:1) donor liposomes are used. In the presence of PC/PE or PC liposomes the exchange activity by this protein is unaffected. In the same experimental conditions, the activity of rat liver non-specific transfer protein is always stimulated by N-ethylmaleimide with all the types of liposomes tested in the order PC/PI greater than PC/PE greater than PC. Since the effect of NEM depends on the type of liposomes used and appears to be similar for both phospholipid transfer proteins, the possibility that their mode of action implies the formation of a ternary complex should be considered. As far as non-specific transfer protein is concerned, its interaction could vary depending on the nature of the exchanging membranes. Data are also presented indicating that when the two transfer proteins are together their activity is additive, therefore suggesting a specific role in phospholipid biomembrane assembly for each of them.

Androgen-Binding Protein↗
PubMed1982

Phenol sulfotransferase. II. Inactivation by phenylglyoxal, N-ethylmaleimide and ribonucleotide 2',3'-dialdehydes.

Phenylglyoxal, a chemical modifying agent for arginine residues, produced rapid inactivation of a rat liver phenol sulfotransferase (3-phosphoadenylylsulfate:phenol sulfotransferase, EC 2.8.2.1). Enzyme inactivation was accompanied by incorporation of 1.5 mol [7-14C]phenylglyoxal per mol enzyme. 3'-Phosphoadenosine 5'-phosphosulfate (PAPS), the sulfate donor, prevented inactivation and decreased [7-14C]phenylglyoxal incorporation to 0.78 mol/mol enzyme. The sulfhydryl-modifying agent, N-ethylmaleimide, also caused rapid inactivation of phenol sulfotransferase with concomitant incorporation of 2.35 mol N-[3H]ethylmaleimide per mol enzyme. These results suggest a possible role for arginine residues as anionic recognition sites for the sulfate donor PAPS, and indicate the presence of essential sulfhydryl residues on phenol sulfotransferase. Ribonucleotide dialdehydes (ATPDA, ADPDA, AMPDA, APSDA), but not the corresponding 2',3'-acyclic nucleotides (ATPDO, ADPDO, AMPDO, APSDO), produced rapid and irreversible inactivation of phenol sulfotransferase. These ribonucleotide dialdehydes appear to modify the active site of the enzyme, since inclusion of the sulfate donor, PAPS, or the product, adenosine 3',5'-bisphosphate (PAP), in the incubation mixture prevented loss of enzyme activity. In contrast, the sulfate acceptor, p-nitrophenol, did not show similar protective effects. Kinetic studies indicated that the ribonucleotide dialdehydes inactivated the enzyme via a unimolecular reaction within a dissociable enzyme-inhibitor complex rather than via a nonspecific bimolecular process. Radioactively labeled ribonucleotide dialdehydes (e.g.,[2, 8-3H]ATP) were incorporated into protein concomitant with loss of enzyme activity. The incorporated ligand could be removed by dialysis in phosphate or Tris buffer. The protein-ligand complex could be stabilized to dialysis by pretreatment with sodium borohydride. The results of these studies suggest that ribonucleotide dialdehydes are affinity labeling reagents for phenol sulfotransferase, causing enzyme inactivation by the possible formation of a Schiff base adduct with an active-site lysine residue.

Aldehydes↗
PubMed1993

N-ethylmaleimide inhibition of silver-enhanced sodium transport across toad (Bufo macinus) skin.

1. Silver stimulated short-circuit current and transepithelial potential difference. 2. N-Ethylmaleimide inhibited the silver-induced short-circuit current. 3. There was a biphasic inhibition of silver-induced short-circuit current by N-ethylmaleimide. 4. There is a specific, maximal number of sulfhydryl groups associated with active sodium absorption. 5. Stimulation of active sodium transport by antidiuretic hormone was blunted by the presence of silver. 6. The silver-induced short-circuit current is carried by a net active sodium transfer from the outside to the inside bathing solution.

Animals↗
PubMed2003

Alkylation of sulfhydryl groups on Galpha(s/olf) subunits by N-ethylmaleimide: regulation by guanine nucleotides.

In rat striatum A(2A) adenosine receptors activate adenylyl cyclase through coupling to G(s)-like proteins, mainly G(olf) that is expressed at high levels in this brain region. In this study we report that the sulfhydryl alkylating reagent, N-ethylmaleimide (NEM), causes a concentration- and time-dependent inhibition of [3H] 2-p-(2-carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamido adenosine ([3H]CGS21680) binding to rat striatal membranes. Membrane treatment with [14C]N-ethylmaleimide ([14C]NEM) labels numerous proteins while addition of 5'-guanylylimidodiphosphate (Gpp(NH)p) reduces labeling of only three protein bands that migrate in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of approximately 52, 45 and 39 kDa, respectively. The 52- and 45-kDa labeled bands show electrophoretic motilities as Galpha(s)-long and Galpha(s)-short/Galpha(olf) subunits. An anti-Galpha(s/olf) antiserum immunoprecipitates two 14C labeled bands of 44 and 39 kDa. The band density decreases by 21-26% when membranes are treated with NEM in the presence of Gpp(NH)p. An anti-A(2A) receptor antibody also immunoprecipitates two 14C labeled bands of 40 and 38 kDa, respectively. However, such protein bands do not show any decrease of their density upon membrane treatment with NEM plus Gpp(NH)p. These results indicate that in rat striatal membranes NEM alkylates sulfhydryl groups of both Galpha(s/olf) subunits and A(2A) adenosine receptors. In addition, cysteine residues of Galpha(s/olf) are easily accessible to modification when the subunit is in the GDP-bound form. The 39- and 38-kDa labeled proteins may represent proteolytic fragments of Galpha(s/olf) and A(2A) adenosine receptor, respectively.

Alkylation↗
PubMed1994

Catalytically distinct conformations of the ribonuclease H of HIV-1 reverse transcriptase by substrate cleavage patterns and inhibition by azidothymidylate and N-ethylmaleimide.

The RNase H activity of recombinant HIV-1 reverse transcriptase (RT) has been characterized with respect to inhibition by azidothymidylate (AZTMP) and N-ethylmaleimide (NEM) and to cleavage patterns using either poly(rA)/poly(dT) or poly(rG)/poly(dC) as model substrate and either Mg2+ or Mn2+ as divalent cation activator. The inhibitory potency of AZTMP and other nucleotide analogues was found to be dependent on both the composition of the substrate and the divalent cation. The enzyme was significantly more sensitive to AZTMP inhibition with poly(rG)/poly(dC) than with poly(rA)/poly(dT) as substrate and in Mn2+ than in Mg2+ with either substrate. Kinetic studies indicated that AZTMP is a competitive inhibitor with respect to the substrate in Mn2+ whereas it behaves as an uncompetitive inhibitor in Mg2+. These results suggest that the enzyme may exist in two distinct forms depending on whether Mg2+ or Mn2+ is the divalent cation activator. Consistent with this suggestion is the alteration in the mode of cleavage of the substrate upon substitution of Mg2+ with Mn2+. In Mg2+, hydrolysis of poly(rA)/poly(dT) appears to be solely endonucleolytic, whereas in Mn2+, hydrolysis is both endonucleolytic and exonucleolytic. With poly(rG)/poly(dC) as substrate, hydrolysis is both endonucleolytic and exonucleolytic in either Mg2+ or Mn2+. There is a positive correlation between sensitivity to AZTMP and production of mononucleotides, suggesting that the exonuclease activity of RNase H is preferentially inhibited by AZTMP. The sensitivity of RNase H to inhibition by N-ethylmaleimide was also found to be markedly influenced by the substrate composition and the divalent cation activator, being most sensitive under conditions in which endonucleolytic activity predominates.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalysis↗
PubMed1986

Purification of labeled cyanogen bromide peptides of the alpha polypeptide from sodium ion and potassium ion activated adenosinetriphosphatase modified with N-[3H]ethylmaleimide.

Sodium ion and potassium ion activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-[3H]ethylmaleimide while it was poised in three different conformations, ostensibly E2-P, E2, and E1, respectively. These assignments were made from a consideration of the particular concentrations of ligands in the respective alkylation mixtures. After a 30-min reaction, the remaining enzymatic activity was found to vary among these three different samples from 90 to 30% of that of unalkylated controls. In all cases, the alpha polypeptide was purified and subjected to digestion with cyanogen bromide, and in each digest the same two distinct radioactive peptides were identified and purified by gel filtration on a column of Sephadex LH-60. The incorporation of N-[3H]ethylmaleimide into one of these two peptides correlated closely with enzymatic inactivation, while the incorporation into the other was most extensive when the portion of the active site to which ATP binds was unoccupied. Alkylation of the residue within the latter peptide, however, does not result in inactivation of the enzyme. Both peptides were further purified by high-pressure liquid chromatography, and their amino-terminal sequences were determined by manual dansyl Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluoresceinyl 5'-isothiocyanate [Farley, R. A., Tran, C. M., Carilli, C. T., Hawke, D., & Shively, J. E. (1984) J. Biol. Chem. 259, 9532-9535].

Amino Acid Sequence↗