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PubMed1985

N-ethylmaleimide as oxidizing agent in biological and non-biological systems.

The hypothesis that N-ethylmaleimide (NEM) may function as an oxidizing agent in biological and non-biological systems, has been tested. Spectrophotometric determination of cytochrome a-redox-state have revealed that NEM promotes the transition of this respiratory chain component in a more oxidated state. To overcome the possibility that the NEM effect may be determined by the inhibition on primary dehydrogenase, duroquinol (QH2) has been used as substrate in the presence of rotenone and malonate. The stimulation of cytochrome a-oxidation is correlated to the one on the QH2 oxidation determined by following the formation of duroquinone. In the absence of any biological system, spontaneous oxidation of ferrocyanide and TMPD is greatly increased by NEM. The differential stimulation induced by maleimide and succinimide indicate that the oxidizing effect of NEM may be considered a chemico-physical property of its molecule mainly due to the presence of a double bond. It is proposed that besides a sulfhydryl reagent, NEM behaves as an oxidizing agent with an interacting site in the region QH2-cytochrome a of the respiratory chain.

Animals↗
PubMed1987

Modulation of alveolar macrophage-derived 5-lipoxygenase products by the sulfhydryl reactant, N-ethylmaleimide.

The sulfhydryl reactant N-ethylmaleimide (NEM) stimulates the release and cyclooxygenase metabolism of arachidonic acid in rat alveolar macrophages. Because both 5-lipoxygenation and leukotriene (LT) C4 synthesis represent sulfhydryl-dependent steps in the 5-lipoxygenase pathway, we examined the effect of NEM on 5-lipoxygenase, as well as cyclooxygenase, metabolism in resting and agonist-stimulated cells by reverse-phase high performance liquid chromatography and radioimmunoassay. NEM at 5-10 microM stimulated the synthesis of thromboxane, but not prostaglandin E2 or the 5-lipoxygenase products LTC4, LTB4, or 5-hydroxyeicosatetraenoic acid from endogenously released arachidonate. In the presence of exogenous fatty acid, however, NEM stimulated the synthesis of large quantities of LTB4. The effect of NEM on arachidonate metabolism stimulated by the calcium ionophore A23187 and the particulate zymosan was also investigated. NEM augmented arachidonate release and thromboxane synthesis stimulated by A23187 but inhibited A23187-induced LTC4 synthesis with an IC50 of approximately 4.3 microM. This inhibitory effect closely paralleled the ability of NEM to deplete intracellular glutathione (IC50 approximately 4.3 microM). Preincubation with the intracellular cysteine delivery agent L-2-oxothiazolidine-4-carboxylate augmented intracellular glutathione concentration and A23187-stimulated LTC4 synthesis and attenuated the capacity of NEM to deplete glutathione and inhibit LTC4 synthesis. While LTB4 and 5-hydroxyeicosatetraenoic synthesis were unaffected at these low NEM concentrations, LTB4 synthesis was inhibited at high concentrations (IC50 approximately 210 microM). Zymosan-induced eicosanoid synthesis was modulated by NEM in a similar fashion. Thus, NEM is an agonist of arachidonate metabolism with the capacity to modulate the spectrum of macrophage-derived eicosanoids by virtue of specific biochemical interactions with substrates and enzymes of the 5-lipoxygenase pathway.

Animals↗
PubMed1987

Effects of N-ethylmaleimide on arginine vasopressin-induced responses in an established smooth muscle cell line.

We have previously reported that 8-arginine vasopressin (AVP) stimulates phosphatidylinositol turnover and Ca++ mobilization in rat aortic smooth muscle cells (A10). In the present study, N-ethylmaleimide (NEM) was used to further characterize the putative guanine nucleotide binding protein that transduces the V1 receptor effects on phosphatidylinositol turnover and Ca++ efflux in these cells. Pretreatment of the cells with low concentrations of NEM did not affect the basal levels of the inositol phosphates and Ca++ efflux but significantly inhibited the AVP-induced increases. NEM pretreatment did not significantly affect [3H]AVP binding to intact cells. Guanyl-5'-yl imidodiphosphate reduced the apparent binding affinity of AVP to cell membranes. NEM pretreatment abolished this guanyl-5'-yl imidodiphosphate effect. AVP stimulated a specific GTPase activity in cell membranes; this effect was also abolished by NEM pretreatment. The results suggest that in A10 cells a guanine nucleotide binding protein sensitive to NEM couples vasopressin receptors to phospholipase C.

Animals↗
PubMed1988

N-ethylmaleimide differentially affects adenosine and thymidine uptake by rat thymocytes.

The involvement of membrane sulfhydryl groups in the uptake of adenosine and thymidine was examined in rat thymocytes pretreated with 6,6'-dithiodinicotinic acid (CPDS) and N-ethylmaleimide (NEM). CPDS, which is known to react uniquely with external membrane sulfhydryls, under short incubation conditions, did not significantly affect the uptake of adenosine and thymidine. Formation of cAMP in nonstimulated and adenosine-stimulated cells was also unaltered by CPDS. However, inhibition of adenosine uptake by competitive inhibitor, dipyridamole, was significantly stronger when the cells were pretreated with CPDS. Preincubation of cells with NEM showed differential sensitivity of adenosine and thymidine uptake, depending on concentration of this sulfhydryl alkylating agent. The results suggest the involvement of NEM-accessible sulfhydryls in membrane transport of adenosine and thymidine. Dual effect of NEM on nucleoside transport may be related to the complexity of nucleoside carrier(s) or to the existence of different nucleoside carriers within thymocyte membranes. On the other hand, the easily accessible, external membrane -SH groups which can be blocked with CPDS, are not essential in thymocyte nucleoside transport but they appear to be situated at a site which interacts with the membrane transport system of nucleoside.

Adenosine↗
PubMed1988

Studies in the rat of antibody-sensitized and N-ethylmaleimide-treated erythrocyte clearance by the liver: effects of immune complex infusion and complement activation.

The function of the hepatic component of the mononuclear phagocyte system (MPS) was investigated in the rat using N-ethylmaleimide (NEM)-treated erythrocytes and erythrocytes sensitized with a rat IgG2a monoclonal antibody (R2/15S) directed against the rat RT1Aa class I antigen. The clearance of the antibody-sensitized erythrocytes was biphasic, with the initial phase being rapid and complement dependent. This clearance was entirely hepatic and could be reduced or prevented by the infusion of cobra venom factor (CVF), complement fragments or small amounts of immune complexes, prepared either at equivalence or 10-fold antigen excess. NEM-treated cells were removed rapidly from the circulation of normal rats (t 1/2 6.2 +/- 0.4 min) and their clearance rate was not influenced by complement depletion. Most of the cells accumulated in the liver, although a small proportion (17%) was removed by the spleen. Immune complexes formed at equivalence or heat-aggregated human gamma globulin (HAGG) delayed the removal of these cells from the circulation, although significantly larger amounts of HAGG were required to do this. Antigen-excess immune complexes had no effect.

Animals↗
PubMed1986

The effects of N-ethylmaleimide on the phosphorylation and aggregation of insulin receptors in the isolated plasma membranes of 3T3-F442A adipocytes.

We have examined the insulin-dependent phosphorylation of the insulin receptor in the isolated plasma membranes of 3T3-F442A adipocytes. Phosphorylation of the insulin receptor is detected readily in the plasma membrane of these cells by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of sodium dodecyl sulfate and under nonreducing conditions, the predominant species of phosphorylated insulin receptor has an apparent molecular mass of 350,000 daltons with the beta-subunit (92,000 daltons) being phosphorylated. The phosphorylation of the insulin receptor in the isolated plasma membrane is absolutely dependent on insulin; tyrosine residues and, to a lesser extent, serine residues of the receptor are phosphorylated. Treatment of the plasma membrane with N-ethylmaleimide (NEM) has two effects; 1) NEM prevents the formation of a larger form (greater than 350,000 daltons) of the phosphorylated insulin receptor. The formation of this larger form of the receptor involves sulfhydryl oxidation which occurs at 37 degrees C under nondenaturing conditions, but does not occur at 0 degrees C or at room temperature even in the presence of sodium dodecyl sulfate. These results indicate that the larger form of the phosphorylated receptor can occur under physiological conditions and suggest that this process may be relevant to aggregation of the receptor-ligand complex. 2) Prior to treatment with insulin, NEM enhances the phosphorylation of the insulin receptor. Phosphopeptide analysis indicates that the site(s) of phosphorylation of the receptor is identical in the presence or absence of NEM.

Adipose Tissue↗
PubMed1986

N-ethylmaleimide cannot inhibit actin polymerization in platelets.

We investigated the effects of the N-ethylmaleimide (NEM), a sulfhydryl(SH) radical blocker, on platelet activation. Platelet aggregation and ATP release was suppressed by 0.2 mM NEM during ADP (20 microM) stimulation and by 0.5 mM NEM during A23187 (4 microM) stimulation. However the agent had no effect on actin polymerization in stimulated platelets. In the absence of a stimulant, NEM (over 1 mM) induced shape changes and slight (5%) actin polymerization, but not aggregation or ATP release. Although platelet aggregation and ATP release were suppressed by the addition of 1 mM NEM during the process of both reactions, the amount of polymerized actin was not influenced by the addition. The reconstructed system consisting of actin and partially purified regulatory proteins without myosin showed a dose-dependent increase in turbidity by the addition of NEM. From these findings, we concluded that NEM enhances actin polymerization, although actin molecules contain SH-radicals, and that actin polymerization has little affect on aggregation and release reaction.

Actins↗
PubMed1986

Studies in the rat of antibody-coated and N-ethylmaleimide-treated erythrocyte clearance by the spleen. I. Effects of in vivo complement activation.

The splenic component of the mononuclear phagocyte system (MPS) was investigated in the rat using N-ethylmaleimide-treated erythrocytes (NEM) and erythrocytes coated with a monoclonal IgG2b antibody (R3/13) directed against the rat RT1Aa major histocompatibility antigen. Both cell suspensions were removed by the spleen, and their clearance times were significantly longer in splenectomized animals. The mean clearance times for the NEM-treated cells in both normal and cobra venom-treated rats were similar (19.1 +/- 1.1 min and 19.0 +/- 1.0 min, respectively) but differences were seen between the clearance of R3/13 antibody-sensitized cells in these two groups (normal rats 38.3 +/- 2.8 min and CVF-treated rats 51.7 +/- 4.2 min, P less than 0.02). Different receptors were also involved in the removal of these cells; in normal animals recognition entailed interaction with complement receptors, whereas in CVF-treated animals this was implemented by Fc receptors. Complement activation prolonged the clearance rates of both R3/13 cells and NEM cells in normal animals, but the effect of complement activation on the clearance of NEM-treated cells was achieved via changes in splenic blood flow. When this was prevented from taking place no effect was seen on the clearance of NEM cells, although the clearance of R3/13 cells was inhibited by the complement fragments generated by complement activation.

Animals↗
PubMed1985

N-ethylmaleimide-induced changes in agonist affinity for histamine H1-receptors in the guinea pig brain.

The effect of the thiol-alkylating agent, N-ethylmaleimide (NEM), on histamine (HA) H1-receptors from guinea pig cerebellum, labeled with [3H]mepyramine, was investigated. The properties of [3H]mepyramine binding (apparent dissociation constant and maximal number of sites) were not modified by prior treatment of the membranes with 2 or 5 mM NEM. This treatment did not change either the inhibition curves of d-chlorpheniramine or mianserin, two H1-receptor antagonists. In contrast, treatments of membranes with NEM significantly decreased the IC50 values of HA and the slope indexes (pseudo Hill coefficients) of HA inhibition curves, which became inferior to unity. These effects were irreversible, and their extent related to the NEM treatment duration and the NEM concentration. A computer analysis of the data indicated that part of the H1-receptors were converted from a state of low affinity for the amine (IC50 value of 75 microM) into a high agonist affinity state (IC50 value of 2 microM). The change was less marked for partial agonists than for HA. The NEM-induced change was observed in the presence and in the absence of Na+ ions, known to decrease the affinity of HA for H1-receptors. Agonists or antagonists did not protect against the modification of HA affinity induced by NEM. The digitonin-solubilized receptors retained their sensitivity to NEM. Among other thiol reagents, iodoacetamide and iodoacetic acid were ineffective, and organic mercurial agents strongly reduced the number of [3H]mepyramine-binding sites. NEM treatment might alkylate a critical thiol group located outside the ligand-binding domain of the H1 receptor and thereby stabilize the latter in a conformation distinct from that of the activated state.

Animals↗
PubMed1984

Interaction of opiate receptor binding sites and guanine nucleotide regulatory sites: selective protection from N-ethylmaleimide.

Guanine nucleotides decrease the binding of opiate agonists to opiate receptors by increasing dissociation rates. To explore the relationship between receptor binding and GTP regulation, rat brain membranes were incubated with N-ethylmaleimide (NEM) and assayed for GTP-induced decrease of 3H-agonist binding. NEM decreased both binding of [3H]dihydromorphine and the effect of GTP on binding. The GTP effect on D-[3H]Ala2-Met5-enkephalinamide (D-Ala-enk) binding was less sensitive to NEM than the binding itself. Incubation of membranes with 0.2 microM D-Ala-enk before addition of NEM significantly protected D-[3H]Ala-enk binding, but did not protect the GTP effect, actually increasing the effect of NEM and eliminating the GTP effect. These "unregulated" receptors were still inhibited by NaCl, but were not stimulated by divalent cations. Although opiate protection alone could not protect the GTP regulation, preincubation of membranes with GTP or guanyl-yl-5'-imidodiphosphate fully protected the GTP effect from NEM and also partially protected the binding site. Preincubation of membranes either with NaCl alone or with NaCl plus D-Ala-enk protected both the GTP effect and the binding site. These results suggest that although the sites for GTP regulation and opiate receptor binding are physically distinct, interactions between sites occur upon the binding of either opiate or guanine nucleotide ligands to their respective sites, and that sodium may play a crucial role in the coupling of these interactions.

Adenylyl Imidodiphosphate↗
PubMed1982

Inhibition of the plasma membrane [H+]-ATPase of Neurospora crassa by N-ethylmaleimide. Protection by nucleotides.

Pre-incubation of the plasma membrane [H+]-ATPase of Neurospora crassa with the sulfhydryl reagent, N-ethylmaleimide (NEM), leads to a marked inhibition of ATPase activity. Loss of activity depends upon preincubation pH and follows pseudo-first order kinetics with respect to NEM concentration. MgATP, the physiological substrate for ATPase activity, protects against NEM inactivation with an average dissociation constant of 1.5 mM at 0 degrees C. This value agrees well with the measured Km for MgATP hydrolysis (1.3 mM at 30 degrees C and 0.9 mM at 15 degrees C). MgADP also protects against NEM inhibition with an average KD of 0.18 mM at 0 degrees C; MgADP is a competitive inhibitor of enzyme activity, with a Ki of 0.08 mM at 30 degrees C and 0.09 mM at 15 degrees C. Free ATP and ADP, as well as other Mg nucleotides (MgGTP, MgCTP, and MgUTP) which are hydrolyzed at much slower rates than MgATP, exert smaller protective effects. These results suggest that nucleotides protect against NEM inhibition by binding to the catalytic site of the ATPase. NEM can therefore be used as a probe to study the nature of enzyme-ligand interactions.

Adenosine Diphosphate↗
PubMed1983

[14C]N-ethylmaleimide labeling of the plasma membrane [H+]-ATPase of Neurospora crassa.

Treatment of the purified plasma membrane [H+]-ATPase of Neurospora crassa with the sulfhydryl reagent N-ethylmaleimide (NEM) leads to a marked inhibition of ATPase activity. Several lines of evidence indicate that inhibition is caused by the reaction of NEM with a single cysteine residue on the Mr = 104,000 polypeptide. (1) Inhibition by NEM follows pseudo-first order kinetics. (2) MgADP protects against NEM inactivation and at the same time decreases the incorporation of [14C]NEM into the Mr = 104,000 polypeptide. When "protectable" [14C]NEM incorporation is plotted as a function of inhibition of ATPase activity, a linear relationship is observed with a slope of 0.8. (3) Labeling of the ATPase with [14C]NEM can be restricted to the protectable site by pretreatment with cold NEM in the presence of MgADP, followed by an incubation with [14C]NEM in the absence of nucleotides. When this is done, and the enzyme is subjected to tryptic mapping and autoradiography, a single radioactive peptide fragment is found. The protectable site may yield information about the role of cysteine in the ATPase mechanism, and should also serve as a useful point of reference in enzyme mapping studies.

Adenosine Triphosphatases↗
PubMed1983

Effects of Mg2+ ions on the plasma membrane [H+]-ATPase of Neurospora crassa. I. Inhibition by N-ethylmaleimide and trypsin.

We have shown previously (Brooker, R.J., and Slayman, C.W. (1982) J. Biol. Chem. 257, 12051-12055; Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 222-226) that the plasma membrane [H+]-ATPase of Neurospora crassa is inhibited by N-ethylmaleimide (NEM), which reacts at an essential nucleotide-protectable site on the Mr = 104,000 polypeptide. The present study demonstrates that Mg2+ has a biphasic effect on NEM inhibition. At low concentrations (0.01-0.1 mM, Mg2+ decreases the sensitivity of the enzyme to NEM, while at high concentrations (greater than 1 mM), it enhances sensitivity. These effects are seen in the presence or absence of nucleotides (ATP, ADP). Mg2+ also acts in a concentration-dependent way to influence the degradation of the ATPase by trypsin. Low concentrations of Mg2+ have little or no effect on tryptic inactivation of ATPase activity or on the disappearance of the Mr = 104,000 polypeptide and the stepwise appearance of Mr = 100,000 and 91,000 tryptic fragments. High concentrations of Mg2+ decrease the rate of inactivation, and a new fragment of Mr = 98,000 is seen. Taken together, the NEM and trypsin results indicate that the Neurospora [H+]-ATPase possesses high and low affinity Mg2+ binding sites which affect the conformation of the enzyme. The divalent cation specificity of the sites has also been investigated. Co2+, Mn2+, and (to a lesser extent) Ni2+ mimic the behavior of Mg2+, but Ca2+ has a different effect, at least at the high affinity site. It appears to bind to that site, based on its ability to inhibit ATP hydrolysis (in the presence of Mg2+), but does not offer protection against NEM inhibition. The results suggest a way in which Ca2+ may serve as a physiological regulator of the ATPase.

Adenosine Diphosphate↗
PubMed1982

Uncoupling of alpha-adrenoceptor-mediated inhibition of human platelet adenylate cyclase by N-ethylmaleimide.

Human platelet adenylate cyclase is stimulated by prostaglandin E1 (PGE1) and is inhibited by epinephrine via alpha-adrenoceptors. Both agonists, epinephrine more than PGE1, increase the activity of a low Km GTPase in platelet membranes. Pretreatment of intact platelets or platelet membranes with the sulfhydryl reagent, N-ethylmaleimide (NEM), abolished the inhibition of the adenylate cyclase and the concomitant stimulation of the GTPase by epinephrine. In contrast, stimulation of the adenylate cyclase by PGE1 was not affected or even increased by NEM pretreatment; only at high NEM concentrations were both basal and PGE1-stimulated activities decreased. Similarly, the PGE1-induced activation of the low Km GTPase was not or was only partially reduced by NEM. Adenylate cyclase activation by stable GTP analogs, NaF, and cholera toxin was also not decreased by NEM pretreatment. Exposure of intact platelets to NEM did not reduce alpha-adrenoceptor number and affinities for agonists and antagonists, as determined by [3H]yohimbine binding in platelet particles. The data indicate that NEM uncouples alpha-adrenoceptor-mediated inhibition of platelet adenylate cyclase, leaving the receptor recognition site and the adenylate cyclase itself relatively intact. Although the effect of NEM may be based on a reaction with the alpha-adrenoceptor site interacting with a coupling component, the selective loss of the adenylate cyclase inhibition together with an even increased stimulation of the enzyme by PGE1 suggests that there are two at least partially distinct regulatory sites involved in opposing hormonal regulations of adenylate cyclase activity, with that involved in hormonal inhibition being highly susceptible to inactivation by NEM.

Adenylyl Cyclases↗
PubMed1983

Protection of opiate receptors in NG108-15 against modification by N-ethylmaleimide.

Two different -SH groups associated with the opiate receptors of the mouse neuroblastoma X rat glioma hybrid NG108-15 have been identified. Modification of these by N-ethylmaleimide (NEM) (presumed to be via alkylation) or by para-chloromercuribenzoic acid (presumed to be via formation of mercury adducts) decreases the binding of both opiate agonists and antagonists to these receptors. Agonist binding is more sensitive than antagonist binding to modification by NEM. Losses in antagonist binding are accounted for totally by decreases in the number of binding sites; there are no corresponding losses in antagonist affinity. Losses of antagonist binding exhibit a pseudo-first order rate constant; the modification of only one such group completely destroys the binding site. Both agonists and antagonists protect against modification of this group by NEM. Sodium and lithium, but not GTP, also protect this group, indicating that the action of these monovalent cations is directly on the receptor moiety. Losses in agonist binding stem not only from decreases in receptor number but also from selective losses in affinity. This -SH group appears to be different from the one at the binding site as sodium, GTP, and antagonist ligands do not protect against losses in agonist affinity. Agonist high affinity also is lost in a pseudo-first order fashion indicating that an alteration of only one -SH group per receptor complex is sufficient to produce this effect. The possible roles of two sulfhydryls in opiate receptor function are discussed.

Animals↗
PubMed1983

N-ethylmaleimide effects on synaptic transmission in frog sympathetic ganglion.

In the isolated bullfrog sympathetic ganglion N-ethylmaleimide (NEM) produced time- and concentration-dependent effects that were irreversible by bathing in drug-free Ringer's solution. Initially, synaptic transmission was facilitated; this was evident as augmented action potential amplitude, repetitive synaptic firing and partial reversal of low [Ca++] transmission failure. Subsequently, transmission was depressed progressively to the point of complete block. Conduction in B and C fiber groups of the preganglionic nerve trunk was also blocked by NEM, without initial facilitation, but at a rate slower than the block of synaptic transmission. NEM had no effect on the amplitude of ganglion cell membrane depolarization produced by acetylcholine, suggesting that its effects were not exerted on postsynaptic nicotinic receptors. None of the effects of NEM were produced by the saturated analog N-ethylsuccinimide. Facilitatory effects of NEM may be attributable to inhibition of Na+ inactivation, or to increased entry of Ca++, at presynaptic nerve terminals. Because the late depressant stages of NEM action involve multiple sites (presynaptic, postsynaptic and axonal), the clarification of possible mechanisms will be a difficult experimental problem.

Animals↗
PubMed1984

Modification of receptor-mediated inhibition of adenylate cyclase in NG108-15 neuroblastoma X glioma cells by n-ethylmaleimide.

Previous studies with membranes from rat heart (Mol. Pharmacol. 21: 570-580, 1982) and human platelets (J. Biol. Chem. 257: 2829-2833, 1982) have suggested that inhibition of adenylate cyclase by occupation of hormone receptors is blocked by pretreatment of membranes with relatively low concentrations of N-ethylmaleimide (NEM). Using membranes derived from NG108-15 neuroblastoma X glioma cells as a model system, we have examined the effect of NEM on the interaction of three inhibitory receptors with adenylate cyclase. Pretreatment of membranes with 100 to 216 microM NEM resulted in a loss of the capacity of agonists to inhibit adenylate cyclase through muscarinic cholinergic and opiate receptors and a loss of GTP-sensitive high-affinity binding of agonists to both of these receptors. Under the same conditions, stimulation of adenylate cyclase by prostaglandin E1 was unchanged. In contrast to the total loss of capacity to inhibit adenylate cyclase by muscarinic and opiate receptor activation, the inhibition of adenylate cyclase by activation of alpha-2 adrenergic receptors was only partially blocked by maximally effective concentrations of NEM. Similarly, GTP-sensitive high-affinity binding of epinephrine to alpha-2 receptors still occurred in NEM (316 microM)-treated membranes. Whereas only a decrease in the efficacy of muscarinic and opiate receptor agonists for inhibition of adenylate cyclase occurred as a result of NEM treatment, pretreatment of membranes with 316 microM NEM resulted in a 30-fold decrease in the potency of epinephrine for inhibition of adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗