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PubMed1997

Survival during exposure to the electrophilic reagent N-ethylmaleimide in Escherichia coli: role of KefB and KefC potassium channels.

The role of the KefB and KefC potassium efflux systems in protecting Escherichia coli cells against the toxic effects of the electrophile N-ethylmaleimide has been investigated. Activation of KefB and KefC aids the survival of cells exposed to high concentrations (> 100 microM) of NEM. High potassium concentrations reduce the protection afforded by activation of KefB and KefC, but the possession of these systems is still important under these conditions. The Kdp system, which confers sensitivity to the electrophile methylglyoxal, did not affect the survival of cells exposed to NEM. Survival is correlated with the reduction of the cytoplasmic pH upon activation of the channels. In particular, the kinetics of the intracellular pH (pHi) change are crucial to the retention of viability of cells exposed to NEM; slow acidification does not protect cells as effectively as rapid lowering of pHi. Cells treated with low levels of NEM (10 microM) recover faster if they activate KefB and KefC, and this correlates with changes in pHi. The pHi does not significantly alter the rate of NEM metabolism. The possible mechanisms by which protection against the electrophile is mediated are discussed.

Antiporters↗
PubMed1998

Importance of RpoS and Dps in survival of exposure of both exponential- and stationary-phase Escherichia coli cells to the electrophile N-ethylmaleimide.

The mechanisms by which Escherichia coli cells survive exposure to the toxic electrophile N-ethylmaleimide (NEM) have been investigated. Stationary-phase E. coli cells were more resistant to NEM than exponential-phase cells. The KefB and KefC systems were found to play an important role in protecting both exponential- and stationary-phase cells against NEM. Additionally, RpoS and the DNA-binding protein Dps aided the survival of both exponential- and stationary-phase cells against NEM. Double mutants lacking both RpoS and Dps and triple mutants deficient in KefB and KefC and either RpoS or Dps had an increased sensitivity to NEM in both exponential- and stationary-phase cells compared to mutants missing only one of these protective mechanisms. Stationary- and exponential-phase cells of a quadruple mutant lacking all four protective systems displayed even greater sensitivity to NEM. These results indicated that protection by the KefB and KefC systems, RpoS and Dps can each occur independently of the other systems. Alterations in the level of RpoS in exponentially growing cells correlated with the degree of NEM sensitivity. Decreasing the level of RpoS by enriching the growth medium enhanced sensitivity to NEM, whereas a mutant lacking the ClpP protease accumulated RpoS and gained high levels of resistance to NEM. A slower-growing E. coli strain was also found to accumulate RpoS and had enhanced resistance to NEM. These data emphasize the multiplicity of pathways involved in protecting E. coli cells against NEM.

Antiporters↗
PubMed1977

Synthesis of Epstein-Barr virus DNA in vitro: effects of phosphonoacetic acid, N-ethylmaleimide, and ATP.

Nuclei from superinfected Raji cells synthesized Epstein-Barr Virus (EBV) DNA in vitro in the absence of cell DNA synthesis. The synthesis of EBV DNA in vitro was inhibited by phosphonoacetic acid and N-ethylmaleimide, and maximum synthesis was achieved in the absence of an ATP-regenerating system. Nuclei from mock-infected cells required an ATP-regenerating system for maximum DNA synthesis.

Acetates↗
PubMed2005

Elimination of retroviral infectivity by N-ethylmaleimide with preservation of functional envelope glycoproteins.

The zinc finger motifs in retroviral nucleocapsid (NC) proteins are essential for viral replication. Disruption of these Cys-X2-Cys-X4-His-X4-Cys zinc-binding structures eliminates infectivity. To determine if N-ethylmaleimide (NEM) can inactivate human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) preparations by alkylating cysteines of NC zinc fingers, we treated infectious virus with NEM and evaluated inactivation of infectivity in cell-based assays. Inactivation was rapid and proportional to the NEM concentration. NEM treatment of HIV-1 or SIV resulted in extensive covalent modification of NC and other internal virion proteins. In contrast, viral envelope glycoproteins, in which the cysteines are disulfide bonded, remained intact and functional, as assayed by high-performance liquid chromatography, fusion-from-without analyses, and dendritic cell capture. Quantitative PCR assays for reverse transcription intermediates showed that NEM and 2,2'-dipyridyl disulfide (aldrithiol-2), a reagent which inactivates retroviruses through oxidation of cysteines in internal virion proteins such as NC, blocked HIV-1 reverse transcription prior to the formation of minus-strand strong-stop products. However, the reverse transcriptase from NEM-treated virions remained active in exogenous template assays, consistent with a role for NC in reverse transcription. Since disruption of NC zinc finger structures by NEM blocks early postentry steps in the retroviral infection cycle, virus preparations with modified NC proteins may be useful as vaccine immunogens and probes of the role of NC in viral replication.

Cell Line↗
PubMedDate not supplied

N-ethylmaleimide-modified actin filaments do not bundle in the presence of alpha-actinin.

We show that the modification of actin subdomain 1 by N-ethylmaleimide (NEM), which binds Cys-374 close to the C-terminus of the molecule, inhibits the alpha-actinin-induced bundling of actin filaments. This effect is not merely related to the block of Cys-374, since N-(1-pyrenyl)iodoacetamide (pyrene-IA) is unable to prevent bundling. Considering that NEM (but not pyrene-IA) influences actin assembly, we suggest that the inhibition of the actin-alpha-actinin interaction is due to the chemical modification of actin Cys-374 which, by inducing a marked spatial reorganization of actin monomers, is able to modify both the intra- and inter-molecular interactions of this protein. Finally, NEM-modified actin filaments form bundles in the presence of polyethylene glycol 6000 since, in this case, the side by side association of actin filaments does not depend on the accessibility of binding sites nor on the formation of chemical bonds.

Actin Cytoskeleton↗
PubMed1985

Stimulation of an N-ethylmaleimide-sensitive ATPase in the collecting duct segments of the rat nephron by metabolic acidosis.

A plasma membrane ATPase sensitive to inhibition by N-ethylmaleimide (NEM) and insensitive to inhibition by oligomycin and ouabain has been shown to be involved in acidification of urine in the turtle bladder. The activity of this NEM-sensitive ATPase was determined in four types of distal nephron segments of normal rats and in rats treated with ammonium chloride. The enzyme activity was determined by a fluorometric micromethod in which ATP hydrolysis was coupled to NADH oxidation. Significant activities (10-35 pmol ADP X min-1 X mm-1) of NEM-sensitive ATPase were present in the distal convoluted tubule (DCT) and in the cortical and outer and inner medullary collecting duct segments of normal rats. In metabolic acidosis produced by ammonium chloride treatment (plasma CO2 content = 15.3 +/- 0.8 mequiv./L), the NEM-sensitive ATPase activity was increased significantly (60-100%) in the collecting duct segments without showing a significant change in the enzyme activity in the DCT. Our data are consistent with the hypothesis that a plasma membrane H+-ATPase (inhibited by NEM but not by oligomycin or ouabain) is involved in H+ secretion in the mammalian collecting duct.

Acidosis↗
PubMed1990

Decrease in N-ethylmaleimide-sensitive ATPase activity in collecting duct by metabolic alkalosis.

Changes in systemic acid-base balance are known to influence acidification in the collecting duct. The H+ secretion in the collecting duct has been shown to be an electrogenic process and it has been suggested that an H-ATPase sensitive to inhibition by N-ethylmaleimide (NEM) is responsible for H+ secretion. This study was designed to determine the effect of metabolic alkalosis on NEM-sensitive ATPase activity in the microdissected segments of the distal nephron. Metabolic alkalosis was produced by giving NaHCO3 to normal rats for 7 days. The plasma total CO2 concentration in the experimental group was 31.5 +/- 1.8 mM compared with 23.4 +/- 1.0 mM in the control group. NEM-sensitive ATPase activity was significantly lower in the cortical collecting duct and in the outer and inner medullary collecting ducts of alkali-loaded rats than those of control rats. There was no significant difference in the enzyme activity between the two groups of animals in the other nephron segments examined. Our results suggest that NEM-sensitive H-APTase activity in all three segments of the collecting duct is modulated by the acid-base status of the animal.

Adenosine Triphosphatases↗
PubMed1981

Locus of N-ethylmaleimide action on sugar transport in nucleated erythrocytes.

Goose red blood cells were studied as a model for metabolic regulation of sugar transport. In contrast to their action in human erythrocytes, sulfhydryl-blocking agents such as N-ethylmaleimide (NEM) stimulated 3-O-methylglucose transport markedly in goose red blood cells. The effect of NEM was further enhanced when adenosine 5'-triphosphate (ATP) was first depleted by 2,4-dinitrophenol treatment or anoxia. Only sulfhydryl-blocking agents that enter the cell were effective transport stimulators, and the effect was not altered by substrates of the transporter. In nucleated red blood cell ghosts, NEM inhibited 3-O-methylglucose transport. Results of these studies with intact cells were consistent with the hypothesis that free sulfhydryl groups are essential for regulation of transporter activity rather than for the transport process itself. The locus of NEM action appears to be either on the cytoplasmic side of the membrane or partially located in the cytoplasm. ATP depletion may expose previously masked sulfhydryl groups, producing an enhanced reaction with sulfhydryl-blocking agents and a highly stimulated rate of sugar transport.

Animals↗
PubMed1984

Activation by N-ethylmaleimide of a latent K+-Cl- flux in human red blood cells.

Twenty to fifty percent of the ouabain-insensitive Na+ and K+ fluxes in human red blood cells are mediated by Cl(-) -dependent coupled transport (cotransport). In this paper we report on the effect of the sulfhydryl group reagent N-ethylmaleimide (NEM) on Cl(-) -dependent ouabain-insensitive Na+ and K+ fluxes in human red blood cells. We found that NEM altered Na+ -K+ cotransport and activated a latent Cl(-) -dependent K+ transport mode normally apparently silent. This conclusion was based on the following observations. 1) At low concentrations (0.25 mM) NEM abolished the bumetanide-sensitive Na+ efflux and had no effect, even at a 10-fold higher concentration, on the bumetanide-sensitive K+ efflux. 2) At concentrations above 0.1 mM, NEM stimulated Cl(-) -dependent K+ efflux that was only partially inhibited by high concentrations of bumetanide or furosemide. In experiments using Rb+ as a K+ analogue, NEM activated Rb+ influx by stimulating the maximum velocity and lowering the apparent external cation affinity. The data suggest the presence of chemically reactive groups in human red blood cells for both Cl(-) -dependent K+ transport activated by NEM and Cl(-) -dependent coupled Na+-K+ movements.

Biological Transport, Active↗
PubMed1987

Effect of N-ethylmaleimide on K transport in density-separated human red blood cells.

N-ethylmaleimide (NEM) is a sulfhydryl-reacting agent known to stimulate chloride-dependent K transport in a variety of red cells. In high K sheep red cells, NEM-induced K movements are greater in magnitude in young cells compared with old cells. We hypothesized that human red cells might respond to NEM like high K sheep red cells. To test this idea, cells of various age were exposed to 0.5 mM NEM. We found that, after a 4-h incubation, young cells lost 50% of cell K, compared with 10% K loss in older cells. K loss in all fractions was inhibited by chloride replacement or furosemide.

Biological Transport↗
PubMed2001

Transcytosis inhibitor N-ethylmaleimide increases microvascular permeability in rat muscle.

N-ethylmaleimide (NEM) has been claimed to markedly inhibit the transvascular passage of small proteins and albumin by interacting with the docking and fusion of plasmalemmal vesicles with their target membranes. To investigate the role of transcytosis in the transcapillary passage of albumin, we assessed the effects of NEM on (125)I-labeled radioiodinated serum albumin clearance (RISA-Cl) from blood to muscle in isolated and maximally vasodilated perfused rat hindquarters, in which vascular pressures, pre- and postcapillary resistances, and the capillary filtration coefficient (CFC) were continuously monitored. NEM (0.3-0.5 mM) caused a marked increase mainly in precapillary vascular resistance. Thus the arterial-to-venous resistance ratio in NEM-treated animals was 3.12 +/- 0.56 versus 1.66 +/- 0.17 during the control period (P < 0.05). Despite that, there was a doubling of both CFC from 0.0363 +/- 0.0028 to 0.0778 +/- 0.0101 ml x min(-1) x mmHg(-1) x 100 g(-1) (P < 0.01) and RISA-Cl, compared with the control situation, signaling markedly increased microvascular permeability. Our results strongly suggest that NEM, besides producing marked vasoconstriction, also causes damage to the capillary endothelium. Thus, instead of inhibiting transvascular transport, NEM may induce increases in the bulk transport of albumin from blood to tissue.

Animals↗
PubMed2004

N-ethylmaleimide causes aquaporin-2 trafficking in the renal inner medullary collecting duct by direct activation of protein kinase A.

The antidiuretic hormone arginine vasopressin increases the osmotic water permeability of the renal collecting ducts by inducing the shuttling of aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical plasma membrane of the principal cells. This process has been demonstrated to be dependent on the cytoskeleton and protein kinase A (PKA). Previous studies in the toad urinary bladder, a functional homologue of the renal collecting duct, have demonstrated that the sulfhydryl reagent N-ethylmaleimide (NEM) is also able to activate the vasopressin-sensitive water permeability pathway in this tissue. The aim of the present study was to investigate the effects of NEM on AQP2 trafficking in a mammalian system. We show that NEM causes translocation of AQP2 from the cytosol to the plasma membrane in rat inner medullary collecting ducts; like the response to arginine vasopressin, this action was also dependent on an intact cytoskeleton and PKA. This effect is not mediated by cAMP but results from direct activation of PKA by NEM.

Animals↗
PubMedDate not supplied

Effects of low-potassium diet on N-ethylmaleimide-sensitive ATPase in the distal nephron segments.

The present study was undertaken to investigate whether or not potassium deficiency influences N-ethylmaleimide (NEM)-sensitive ATPase in the distal nephron segments of the rat. One group of animals was fed a low-K diet, whereas the normal K-group was given the same diet after supplementation with KCl. The nephron segments examined were: the medullary and cortical thick ascending limbs, the distal convoluted tubule, and the cortical, outer and inner medullary collecting ducts. NEM-sensitive ATPase activity in microdissected segments was measured by a fluorometric microassay. The plasma K+ concentration in the low-K group was 3.1 +/- 0.3 mEq/l compared with 4.2 +/- 0.1 mEq/l in the normal-K group. NEM-sensitive ATPase activity in the outer medullary collecting duct of low-K diet animals was significantly greater than in normal-K animals. There was no significant difference in NEM-sensitive ATPase activity between the two groups of animals in the other nephron segments examined. It is suggested that NEM-sensitive H-ATPase activity in the outer medullary collecting duct is modulated by the potassium status of the animal.

Animals↗
PubMed1976

Enzymatic properties of native and N-ethylmaleimide-modified cardiac myosin from normal and thyrotoxic rabbits.

Cardiac myosin from thyrotoxic animals (myosin-T) exhibits elevated Ca2+ -ATPase activity which is resistant to further stimulation by sulfhydryl modification. In the present study, we have compared the enzymatic properties of myosin-T with those of myosin from euthyroid rabbits (myosin-N) and the derivatives of myosin-T and myosin-N formed by blocking the most rapidly reacting class of thiols (SH1) with N-ethylmaleimide (NEM). Vmax for Ca2+ -ATPase of myosin-T was about 250% greater than myosin-N and was nearly the same as NEM-modified myosin-N. Values for the apparent Km of myosin-T and NEM-modified myosin-N were 200% greater than the value for unmodified myosin-N. Vmax and Km for K+ (EDTA)-ATPase activity of NEM-modified myosin-T and myosin-N were identical. The Ca2+ saturation, pH, and salt-dependency curves for the ATPase activity of myosin-T were parallel to the curves for myosin-N and differed from those for the NEM-modified myosins. Myosin-T exhibited an increased rate of hydrolysis of ATP, CTP, and UTP in both low (0.05m) and high (0.5m) KCl medium. NEM-modified myosin-N showed increased hydrolysis of ATP and CTP in low KCl medium and increased hydrolysis of ATP, CTP, and UTP in high KCl medium. These results support the hypothesis that the enzymatic behavior of myosin-T may be caused by an alteration in the active site near the SH, thiols. The unique enzymatic properties of myosin-T did not seem to be the result of a major change in structure. The electrophoretic pattern of light chains from myosin-T and myosin-N was the same in polyacrylamide gels containing either 8 M urea at pH 8.6 or sodium dodecyl sulfate. Also, myosin-T had a normal amino acid composition and lacked 3-methyl-histidine and hot acid-stable phosphate.

Adenosine Triphosphatases↗
PubMed1977

Actin-activated adenosine triphosphatase activity of native and N-ethylmaleimide-modified cardiac myosin from normal and thyrotoxic rabbits.

The Ca2+-ATPase activity of cardiac myosin is increased in thyrotoxic animals. However, the physiological significance of this observation is uncertain since, in living muscle, Mg-ATP is hydrolyzed by myosin under the stimulating influence of actin. In this study, we have compared the actin-activated ATPase activity of myosin from euthyroid (myosin-N) and thyrotoxic (myosin-T) rabbits and the derivatives of myosin-N and myosin-T formed by blocking the most rapidly reacting class of thiols (SH1) with N-ethylmaleimide (NEM). Also, we have studied the activity of these myosins in the presence of a complex of troponin and tropomyosin that confers calcium sensitivity on the system. Vmax for the actin-activated ATPase of myosin-T was about 168% greater than for myosin N. The apparent dissociation constant for actin, Kapp, for myosin-T was about 42% of the normal value. After NEM modification, Vmax and Kapp for NEM-modified myosin-T and myosin-N decreased, becoming essentially the same for both myosins. In the presence of troponin-tropomyosin complex, the actin-activated ATPase of myosin-T exhibited calcium sensitivity that could be reduced by thiol modification. These results suggest that the SH1 thiols or the region near these groups are important to the actin-activated ATPase of myosin-N and are essential to the increased activity of myosin-T. Also, they suggest that the changes in the enzymatic properties of myosin induced by thyroxine may be responsible for altering the contractile properties of the heart.

Actins↗
PubMed1997

N-ethylmaleimide (NEM) can significantly improve in situ hybridization results using 35S-labeled oligodeoxynucleotide or complementary RNA probes.

We predicted that a significant source of background labeling after in situ hybridization (ISH) using 35S-labeled probes is attributable to a chemical reaction between the phosphorothioate moiety of the probe [O3P = S] and disulfides in tissue. These covalent bonds would immobilize probe in the tissue, thereby increasing background labeling. On the basis of this view, we have explored the use of N-ethylmaleimide (NEM) to irreversibly alkylate the phosphorothioate moiety of the probe and/or to alkylate free sulfhydryls in tissue to block the formation of disulfides as a method of reducing background labeling. We report that NEM can significantly decrease background labeling of 35S-labeled oligodeoxynucleotide or cRNA probes but does not affect specific labeling. We conclude that the use of NEM in ISH protocols, as outlined here, may be an additional element researchers may consider to improve the signal-to-noise ratio.

Brain Chemistry↗
PubMed1986

Effects of N-ethylmaleimide on gonadotropin and beta-adrenergic receptor function coupled to rabbit luteal adenylyl cyclase.

The effects of the sulfhydryl-reactive alkylating agent N-ethylmaleimide (NEM) on the rabbit luteal adenylyl cyclase system were studied. Treatment of luteal membranes with NEM revealed three activities with differing sensitivities to NEM treatment. When luteal membranes were treated with NEM on ice for 30 min, it was found that NaF-stimulated adenylyl cyclase activity in cholate extracts of these membranes was most sensitive to this treatment. Half-maximal inhibition was obtained at 0.09 mM NEM. The activity of the stimulatory guanine nucleotide- and Mg-binding regulatory component (Ns), as assessed by functional reconstitution of NaF-stimulated adenylyl cyclase activity into membranes from the cyc-variant of the S49 mouse lymphoma, was less sensitive to this treatment, with half-maximal inhibition occurring at 0.69 mM NEM. In contrast, high affinity gonadotropin and beta-adrenergic binding, as assessed by competitive displacement of [125I]iodo-hCG by bovine LH and (-)3-[125I]iodocyanopindolol by isoproterenol, was unaffected by NEM concentrations up to 50 mM when membranes were treated on ice. However, when membranes were treated with NEM at 25 C for 30 min, high affinity gonadotropin and beta-adrenergic binding demonstrated similar sensitivities to NEM treatment, such that 50 mM NEM completely inhibited high affinity binding to both receptors. Under either of the conditions described above, neither the number of receptors nor the affinities of the labeled probes for their receptors were altered by NEM treatment. Thus, there appears to be at least three NEM-sensitive sites necessary for the functioning of the rabbit luteal adenylyl cyclase system, one associated with the catalytic component, one on Ns which interacts with the catalytic component, and one involved in high affinity agonist binding. Furthermore, it appears that formation of the high affinity binding state is regulated similarly for gonadotropin and beta-adrenergic receptors.

Adenylyl Cyclase Inhibitors↗
PubMed1988

Hepatic vasopressin receptor: differential effects of divalent cations, guanine nucleotides, and N-ethylmaleimide on agonist and antagonist interactions with the V1 subtype receptor.

Previously, we reported that magnesium (Mg2+) enhanced the binding affinity of arginine vasopressin [( 3H]AVP) to a single class of sites in rat liver microsomes. In the present study we have examined the effects of divalent cations and guanine nucleotides on the binding characteristics of both the nonselective agonist and the V1 receptor-selective antagonist, d(CH2)5Tyr(Me)-[3H]AVP, to microsomal and plasma membrane fractions of rat liver. At a subsaturating concentration (100 pM) of [3H]AVP, divalent cations increased specific binding in a concentration-dependent manner with the following rank order of potency: Co2+ greater than Mn2+ greater than Ni2+ greater than Mg2+ greater than Ca2+ = control. The maximal effect for Mg2+ was evident at 1 mM, a physiologically relevant concentration. In contrast, binding of the V1 receptor antagonist (at a subsaturating concentration of 10 pM) was inhibited by divalent cations, the rank order of potency being Mn2+ greater than Co2+ greater than Ca2+ greater than Mg2+ greater than Ni2+. The inhibitory effects of divalent cations were of lesser magnitude (up to 60%) compared to the stimulation of agonist binding (up to 700%). Mg2+ enhanced the affinity of [3H]AVP (Kd was decreased from approximately 2 nM to 133 pM), while the affinity of the [3H]V1 antagonist was decreased (Kd was increased from 10 to 95 pM). Scatchard analysis of saturation data (Mg2+ present) revealed similar maximum binding values for the binding of radiolabeled agonist and antagonist, indicating that AVP receptors in rat liver are mostly of the V1 subtype. Competition experiments between V1/V2-specific AVP analogs with either the radiolabeled agonist or antagonist also indicated the presence of predominantly V1 receptor sites in rat liver microsomes. The properties of plasma membrane receptor sites were similar to those of the microsomal sites, except that the density of receptors was higher in the former. In both equilibrium and competitive inhibition experiments GTPase-resistant analogs of guanine nucleotides, GTP gamma S and GDP beta S, decreased the affinity of the agonist for the receptor, but not that of the antagonist. Treatment of membranes with 0.2 mM N-ethylmaleimide (NEM) reduced the maximum binding of [3H]AVP and abolished the GTP gamma S-evoked decrease in agonist-binding affinity. In contrast, antagonist binding was unaffected by NEM. NEM pretreatment failed to influence the divalent cation-dependent increase in agonist-binding affinity. The results provide direct evidence for the existence of a high and a low affinity state of the hepatic V1 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗