Effects Of N-ethylmaleimide Research Articles

Putative role of intracellular Zn2+ release during oxidative stress: a trigger to restore cellular thiol content that is decreased by oxidative stress

Although the ability of zinc to retard the oxidative process has been recognized for many years, zinc itself has been reported to induce oxidative stress. In order to give some insights into elucidating the role of intracellular Zn(2+) in cells suffering from oxidative stress, the effects of N-ethylmaleimide (NEM) and ZnCl(2) on cellular thiol content and intracellular Zn(2+) concentration were studied by use of 5-chloromethylfluorescein diacetate (5-CMF-DA) and FluoZin-3 pentaacetoxymethyl ester (FluoZin-3-AM) in rat thymocytes. The treatment of cells with NEM attenuated 5-CMF fluorescence and augmented FluoZin-3 fluorescence in a dose-dependent manner. These NEM-induced phenomena were observed under external Zn(2+)-free conditions. Results suggest that NEM decreases cellular thiol content and induces intracellular Zn(2+) release. Micromolar ZnCl(2) dose-dependently augmented both FluoZin-3 and 5-CMF fluorescences, suggesting that the elevation of intracellular Zn(2+) concentration increases cellular thiol content. Taken together, it is hypothesized that intracellular Zn(2+) release during oxidative stress is a trigger to restore cellular thiol content that is decreased by oxidative stress.

Optimal metabolic regulation of the mammalian heart Na +/Ca 2+ exchanger requires a spacial arrangements with a PtdIns(4)-5kinase

In inside-out bovine heart sarcolemmal vesicles, p-chloromercuribenzenesulfonate (PCMBS) and n-ethylmaleimide (NEM) fully inhibited MgATP up-regulation of the Na +/Ca 2+ exchanger (NCX1) and abolished the MgATP-dependent PtdIns-4,5P2 increase in the NCX1–PtdIns-4,5P2 complex; in addition, these compounds markedly reduced the activity of the PtdIns(4)-5kinase. After PCMBS or NEM treatment, addition of dithiothreitol (DTT) restored a large fraction of the MgATP stimulation of the exchange fluxes and almost fully restored PtdIns(4)-5kinase activity; however, in contrast to PCMBS, the effects of NEM did not seem related to the alkylation of protein SH groups. By itself DTT had no effect on the synthesis of PtdIns-4,5P2 but affected MgATP stimulation of NCX1: moderate inhibition at 1 mM MgATP and 1 μM Ca 2+ and full inhibition at 0.25 mM MgATP and 0.2 μM Ca 2+. In addition, DDT prevented coimmunoprecipitation of NCX1 and PtdIns(4)-5kinase. These results indicate that, for a proper MgATP up-regulation of NCX1, the enzyme responsible for PtdIns-4,5P2 synthesis must be (i) functionally competent and (ii) set in the NCX1 microenvironment closely associated to the exchanger. This kind of supramolecular structure is needed to optimize binding of the newly synthesized PtdIns-4,5P2 to its target region in the exchanger protein.

The thiol-modifying agent N-ethylmaleimide elevates the cytosolic concentration of free Zn2+ but not of Ca2+ in murine cortical neurons

The membrane permeant alkylating agent N-ethylmaleimide (NEM) regulates numerous biological processes by reacting with thiol groups. Among other actions, NEM influences the cytosolic concentration of free Ca2+ ([Ca2+]i). Depending on the cell type and the concentration used, NEM can promote the release of Ca2+, affect its extrusion, stimulate or block its entry. However, most of these findings were obtained in experiments that employed fluorescent Ca2+ probes and one major disadvantage of such experimental setting derives from the lack of specificity of the probes as all the so-called “Ca2+-sensitive” indicators also bind metals like Zn2+ or Mn2+ with higher affinities than Ca2+. In this study, we examined the effects of NEM on the [Ca2+]i homeostasis of murine cortical neurons. We performed live-cell Ca2+ and Zn2+ imaging experiments using the fluorescent probes Fluo-4, FluoZin-3 and RhodZin-3 and found that NEM does not affect the neuronal [Ca2+]i homeostasis but specifically increases the cytosolic and mitochondrial concentration of free Zn2+([Zn2+]i). In addition, NEM triggers some neuronal loss that is prevented by anti-oxidants such as N-acetylcysteine or glutathione. NEM-induced toxicity is dependent on changes in [Zn2+]i levels as chelation of the cation with the cell-permeable heavy metal chelator, N,N,N′N′-tetrakis(−)[2-pyridylmethyl]-ethylenediamine (TPEN), promotes neuroprotection of cortical neurons exposed to NEM. Our data indicate that NEM affects [Zn2+]i but not [Ca2+]i homeostasis and shed new light on the physiological actions of this alkylating agent on central nervous system neurons.

Characterization of mENT1Δ11, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1

Mammalian cells require specific transport mechanisms for the cellular uptake and release of endogenous nucleosides such as adenosine, and nucleoside analogs used in chemotherapy. We have identified a novel splice variant of the mouse equilibrative nucleoside transporter, mENT1, that results from the exclusion of exon 11 during pre-RNA processing. This variant encodes a truncated protein (mENT1Delta11) missing the last three transmembrane domains of the full-length mENT1. The mENT1Delta11 transcript and protein were found to be differentially distributed among tissues relative to full-length mENT1. PK15-NTD (nucleoside transport deficient) cells were transfected with mENT1 or mENT1Delta11 and assessed for nucleoside transport function. No significant differences were observed between the mENT1 and mENT1Delta11 in terms of transport function or inhibitor binding affinity. PK15-mENT1Delta11 transfected cells bound the ENT1 probe [3H]nitrobenzylthioinosine (NBMPR) with high affinity and mediated the cellular accumulation of both [3H]2-chloroadenosine and [3H]uridine. The only significant differences between the mENT1 variants were that mENT1Delta11 could not be photolabeled with [3H]NBMPR and that mENT1Delta11 was insensitive to the transporter-modifying effects of N-ethylmaleimide. These data suggest that the last three transmembrane domains of mENT1 are not necessary for transport activity, but this region does contain the cysteines responsible for the sensitivity of mENT1 to sulfhydryl reagents, and the residues important for covalent modification of the protein with NBMPR. These results provide important guidelines for future mutagenesis studies aimed at elucidating the tertiary structure of the ENT1 protein and the domains involved in inhibitor binding and substrate translocation.

Substrate Modification with Lysine 63-linked Ubiquitin Chains through the UBC13-UEV1A Ubiquitin-conjugating Enzyme

Protein modification with lysine 63-linked ubiquitin chains has been implicated in the non-proteolytic regulation of signaling pathways. To understand the molecular mechanisms underlying this process, we have developed an in vitro system to examine the activity of the ubiquitin-conjugating enzyme UBC13-UEV1A with TRAF6 in which TRAF6 serves as both a ubiquitin ligase and substrate for modification. Although TRAF6 potently stimulates the activity of UBC13-UEV1A to synthesize ubiquitin chains, it is not appreciably ubiquitinated. We have determined that the presentation of Lys(63) of ubiquitin by UEV1A suppresses TRAF6 modification. Based on our observations, we propose that the modification of proteins with Lys(63)-linked ubiquitin chains occurs through a UEV1A-independent substrate modification and UEV1A-dependent Lys(63)-linked ubiquitin chain synthesis mechanism.

Two Novel Ubiquitin-fold Modifier 1 (Ufm1)-specific Proteases, UfSP1 and UfSP2

Ubiquitin-fold modifier 1 (Ufm1) is a recently identified new ubiquitin-like protein, whose tertiary structure displays a striking resemblance to ubiquitin. Similar to ubiquitin, it has a Gly residue conserved across species at the C-terminal region with extensions of various amino acid sequences that need to be processed in vivo prior to conjugation to target proteins. Here we report the isolation, cloning, and characterization of two novel mouse Ufm1-specific proteases, named UfSP1 and UfSP2. UfSP1 and UfSP2 are composed of 217 and 461 amino acids, respectively, and they have no sequence homology with previously known proteases. UfSP2 is present in most, if not all, of multicellular organisms including plant, nematode, fly, and mammal, whereas UfSP1 could not be found in plant and nematode upon data base search. UfSP1 and UfSP2 cleaved the C-terminal extension of Ufm1 but not that of ubiquitin or other ubiquitin-like proteins, such as SUMO-1 and ISG15. Both were also capable of releasing Ufm1 from Ufm1-conjugated cellular proteins. They were sensitive to inhibition by sulfhydryl-blocking agents, such as N-ethylmaleimide, and their active site Cys could be labeled with Ufm1-vinylmethylester. Moreover, replacement of the conserved Cys residue by Ser resulted in a complete loss of the UfSP1 and UfSP2 activities. These results indicate that UfSP1 and UfSP2 are novel thiol proteases that specifically process the C terminus of Ufm1.

N‐ethylmaleimide increases release probability at GABAergic synapses in layer I of the mouse visual cortex

The sulphydryl alkylating agent N-ethylmaleimide (NEM) has been often used as an uncoupler of pertussis toxin-sensitive G-proteins. However, the effects of NEM on gamma-aminobutyric acid (GABA)ergic synaptic transmission remain controversial. Using the whole-cell patch-clamp technique, GABA(A) receptor-mediated postsynaptic currents (IPSCs) have been recorded from Cajal-Retzius (CR) cells in layer I of the neonatal mouse visual cortex. NEM increased the frequencies of both spontaneous and miniature IPSCs (mIPSCs) without an effect on the median mIPSC amplitudes or mIPSC kinetics. The NEM actions on mIPSCs did not depend on the extracellular Ca(2+), Ca(2+) release from intracellular stores, adenylyl cyclase and protein kinase A activities. NEM increased the mean amplitudes of evoked IPSCs and strongly decreased the paired-pulse ratio. The size of the readily releasable pool of presynaptic vesicles (RRP) was estimated using a high-frequency stimulation protocol. The RRP size was not affected by NEM. In addition, NEM significantly decreased the latency between the stimulus and the onset of GABA release. These results suggest that NEM selectively increases GABA release probability. At postnatal day 2, mIPSCs were observed only in about 30% of CR cells. NEM application revealed, however, that more than 90% of CR cells receive GABAergic inputs. Therefore, NEM seems to be a useful tool to verify the existence of 'silent' GABAergic synapses.

Characterization ofl-arginine transport in adrenal cells: effect of ACTH

Nitric oxide synthesis depends on the availability of its precursor L-arginine, which could be regulated by the presence of a specific uptake system. In the present report, the characterization of the L-arginine transport system in mouse adrenal Y1 cells was performed. L-arginine transport was mediated by the cationic/neutral amino acid transport system y+L and the cationic amino acid transporter (CAT) y+ in Y1 cells. These Na+-independent transporters were identified by their selectivity for neutral amino acids in both the presence and absence of Na+ and by the effect of N-ethylmaleimide. Transport data correlated to expression of genes encoding for CAT-1, CAT-2, CD-98, and y+LAT-2. A similar expression profile was detected in rat adrenal zona fasciculata. In addition, cationic amino acid uptake in Y1 cells was upregulated by ACTH and/or cAMP with a concomitant increase in nitric oxide (NO) production.

Role of Transmembrane Segment 10 in Efflux Mediated by the Staphylococcal Multidrug Transport Protein QacA

The staphylococcal multidrug exporter QacA confers resistance to a wide range of structurally dissimilar monovalent and bivalent cationic antimicrobial compounds. To understand the functional importance of transmembrane segment 10, which is thought to be involved in substrate binding, cysteine-scanning mutagenesis was performed in which 35 amino acid residues in the putative transmembrane helix and its flanking regions were replaced in turn with cysteine. Solvent accessibility analysis of the introduced cysteine residues using fluorescein maleimide indicated that transmembrane segment 10 of QacA contains a 20-amino-acid hydrophobic core and may extend from Pro-309 to Ala-334. Phenotypic analysis and fluorimetric transport assays of these mutants showed that Gly-313 is important for the efflux of both monovalent and bivalent cationic substrates, whereas Asp-323 is only important for the efflux of bivalent substrates and probably forms part of the bivalent substrate-binding site(s) together with Met-319. Furthermore, the effects of N-ethyl-maleimide treatment on ethidium and 4',6-diamidino-2-phenylindole export mediated by the QacA mutants suggest that the face of transmembrane segment 10 that contains Asp-323 may also be close to the monovalent substrate-binding site(s), making this helix an integral component of the QacA multidrug-binding pocket.

The Relaxant Activity of 4,7-Dimethyl-1,2,5-oxadiazolo[3,4-d]-pyridazine 1,5,6-Trioxide in the Mouse Corpus Cavernosum

We have studied the effect of an activator of soluble guanylate cyclase 4,7-dimethyl-1,2,5-oxadiazolo[3,4-d]pyridazine 1,5,6-trioxide (FPTO) on the tone and nitrergic relaxation responses of mouse cavernous strips and compared FPTO to a known nitric oxide donor sodium nitroprusside. FPTO thiol-dependently generated nitric oxide measured by polarography and activated purified human soluble guanylate cyclase. FPTO and sodium nitroprusside relaxed the cavernous tissue in a concentration-dependent manner. A nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine did not alter the relaxations to FPTO or sodium nitroprusside, whereas soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) suppressed relaxation to FPTO and sodium nitroprusside. Exogenously added thiols L-cysteine or dithiothreitol inhibited the relaxant responses to FPTO but not to sodium nitroprusside, whereas glutathione did not influence the responses to both agents. Thiol alkylation agent N-ethylmaleimide significantly enhanced FPTO-induced relaxation, and thiol-modifying agent diamide inhibited relaxation to FPTO. The potentiating effect of N-ethylmaleimide was neutralized by coadministration of N-ethylmaleimide with glutathione, L-cysteine, dithiothreitol, or ODQ. N-Ethylmaleimide but not diamide significantly inhibited relaxation induced by sodium nitroprusside. FPTO potently suppressed contraction to electrical field stimulation, which was prevented by glutathione or L-cysteine. In addition, FPTO did not affect relaxation produced by electrical field stimulation in phenylephrine-precontracted tissue. Our results show that FPTO can relax mouse corpus cavernosum and that the relaxant activity of this agent is thiol- and soluble guanylate cyclase-dependent. This effect could be potentiated by N-ethylmaleimide. FPTO does not potentiate nitrergic relaxation induced by electrical field stimulation.

Enhanced Dephosphorylation of cAMP-dependent Protein Kinase by Oxidation and Thiol Modification

The catalytic subunit of cAMP-dependent protein kinase (PKA) is phosphorylated at threonine 197 and serine 338. Phosphorylation of threonine 197, located in the activation loop, is required for coordinating the active site conformation and optimal enzymatic activity. However, this phosphorylation has not been widely appreciated as a regulatory site because of the apparent constitutive nature of the phosphorylation and the general resistance of the kinase to phosphatase treatment. We demonstrate here that the observed resistance of the catalytic subunit to dephosphorylation is due, in part, to the presence of the highly nucleophilic cysteine 199 located proximal to the phosphate on threonine 197. Experiments performed in vitro demonstrated that mutation (cysteine 199 to alanine), oxidation, such as by glutathionylation or internal disulfide bond formation, or alkylation of the C-subunit enhanced its ability to be dephosphorylated. Furthermore, rephosphorylation of reduced C-subunit by PDK1 created a cycle whereby the inactive kinase could be reactivated. To demonstrate that thiol modification of PKA can lead to enhanced dephosphorylation in vivo, PC12 cells were treated with N-ethylmaleimide (NEM). Such treatment resulted in complete PKA inactivation and dephosphorylation of threonine 197. This effect of NEM was contingent upon prior treatment of the cells with PKA activators, demonstrating the resistance of the holoenzyme to thiol alkylation-mediated dephosphorylation. Our results also demonstrated that NEM treatment of PC12 cells enhanced the dephosphorylation of the protein kinase Calpha activation loop, suggesting a common mechanism of regulation among members of the AGC family of kinases.

Pronociceptive role of dynorphins in uninjured animals: N-ethylmaleimide-induced nociceptive behavior mediated through inhibition of dynorphin degradation

Intrathecal (i.t.) administration into mice of N-ethylmaleimide (NEM), a cysteine protease inhibitor, produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by NEM was inhibited by the intraperitoneal injection of morphine. We have recently reported that dynorphin A and, more potently big dynorphin, consisting of dynorphins A and B, produce the same type of nociceptive response whereas dynorphin B does not [Tan-No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K. Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl- d-aspartate receptor mechanism. Brain Res 2002;952:7–14]. The NEM-induced nociceptive behavior was inhibited by pretreatment with dynorphin A- or dynorphin B-antiserum and each antiserum also reduced the nociceptive effects of i.t.-injected synthetic big dynorphin. The characteristic NEM-evoked response was not observed in prodynorphin knockout mice. Naloxone, an opioid receptor antagonist, had no effects on the NEM-induced behavior. Ifenprodil, arcaine and agmatine, antagonists at the polyamine recognition site on the N-methyl- d-aspartate (NMDA) receptor ion-channel complex, and MK-801, an NMDA ion-channel blocker inhibited the NEM-induced effects. Ro25-6981, an antagonist of the NMDA receptor subtype containing NR2B subunit was not active. NEM completely inhibited degradation of dynorphin A by soluble and particulate fractions of mouse spinal cord. Collectively, the results demonstrate that endogenous prodynorphin-derived peptides are pronociceptive in uninjured animals, and required for the NEM-induced behavior. The NEM effects may be mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site.

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.

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.

Antibodies and a cysteine-modifying reagent show correspondence of M current in neurons to KCNQ2 and KCNQ3 K+ channels.

1. KCNQ K(+) channels are thought to underlie the M current of neurons. To probe if the KCNQ2 and KCNQ3 subtypes underlie the M current of rat superior cervical ganglia (SCG) neurons and of hippocampus, we raised specific antibodies against them and also used the cysteine-alkylating agent N-ethylmaleimide (NEM) as an additional probe of subunit composition. 2. Tested on tsA-201 (tsA) cells transfected with cloned KCNQ1-5 subunits, our antibodies showed high affinity and selectivity for the appropriate subtype. The antibodies immunostained SCG neurons and hippocampal sections at levels similar to those for channels expressed in tsA cells, indicating that KCNQ2 and KCNQ3 are present in SCG and hippocampal neurons. Some hippocampal regions contained only KCNQ2 or KCNQ3 subunits, suggesting the presence of M currents produced by channels other than KCNQ2/3 heteromultimers. 3. We found that NEM augmented M currents in SCG neurons and KCNQ2/3 currents in tsA cells via strong voltage-independent and modest voltage-dependent actions. Expression of individual KCNQ subunits in tsA cells revealed voltage-independent augmentation of KCNQ2, but not KCNQ1 nor KCNQ3, currents by NEM indicating that this action on SCG M currents likely localizes to KCNQ2. Much of the voltage-independent action is lost after the C242T mutation in KCNQ2. 4. The correspondence of NEM effects on expressed KCNQ2/3 and SCG M currents, along with the antibody labelling, provide further evidence that KCNQ2 and KCNQ3 subunits strongly contribute to the M current of neurons. The site of NEM action may be important for treatment of diseases caused by under-expression of these channels.

Complex effects of sulfhydryl reagents on ligand interactions with nucleoside transporters: evidence for multiple populations of ENT1 transporters with differential sensitivities to N-ethylmaleimide

Functional studies have implicated cysteines in the interaction of ligands with the ENT1 nucleoside transporter. To better define these interactions, N-ethylmaleimide (NEM) and p-chloromercuribenzylsulfonate ( pCMBS) were tested for their effects on ligand interactions with the [ 3 H] nitrobenzylthioinosine (NBMPR) binding site of the ENT1 transporters of mouse Ehrlich ascites cells and human erythrocytes. NEM had biphasic, concentration-dependent effects on NBMPR binding to intact Ehrlich cells, plasma membranes, and detergent-solubilized membranes, with about 35% of the binding activity being relatively insensitive to NEM inhibition. NBMPR binding to human erythrocyte membranes also displayed heterogeneity in that about 33% of the NBMPR binding sites remained, albeit with lower affinity for NBMPR, even after treatment with NEM at concentrations in excess of 1 mM. However, unlike that seen for Ehrlich cells, no “reversal” in NBMPR binding to human erythrocyte membranes was observed at the higher concentrations of NEM. pCMBS inhibited 100% of the NBMPR binding to both Ehrlich cell and human erythrocyte membranes, but had no effect on the binding of NBMPR to intact cells. The effects of NEM on NBMPR binding could be prevented by coincubation of membranes with nonradiolabeled NBMPR, adenosine, or uridine. Treatment with NEM and pCMBS also decreased the affinity of other nucleoside transport inhibitors for the NBMPR binding site, but enhanced the affinities of nucleoside substrates. These data support the existence of at least two populations of ENT1 in both erythrocyte and Ehrlich cell membranes with differential sensitivities to NEM. The interaction of NEM with the mouse ENT1 protein may also involve additional sulphydryl groups not present in the human ENT1.

The sulphydryl reagent, N-ethylmaleimide, disrupts sleep and blocks A1 adenosine receptor-mediated inhibition of intracellular calcium signaling in the in vitro ventromedial preoptic nucleus

To explore the neuronal signaling mechanisms underlying sleep regulation in the rat, the present study examined continuous intra-third ventricle infusion of N-ethylmaleimide (NEM), a sulphydryl reagent that inhibits G i/o protein-coupled receptor-mediated signaling pathways. The diurnal infusion of NEM (0.01–10 μmol/10 h) dose-dependently inhibited both non-rapid eye movement sleep and rapid eye movement sleep. A maximal dose of NEM (10 μmol/10 h) dramatically inhibited day-time sleep (−57% for non-rapid eye movement sleep and −89% for rapid eye movement sleep) with a compensatory increase of sleep during the subsequent night-time (+33% for non-rapid eye movement sleep and +259% for rapid eye movement sleep). The day-time brain temperature was also increased by NEM, demonstrating effects of NEM on both sleep and body temperature levels. Immunostaining of the rat hypothalamus with a monoclonal antibody against the A1 adenosine receptor (A1R) was used to explore the distribution of a sleep-related G i/o protein-coupled receptor. Robust A1R-like immunoreactivity was found in the ventromedial preoptic nucleus and the supraoptic nucleus. Fura-2-based Ca 2+ imaging analysis of acute hypothalamic slices further demonstrated that the A1R agonist N 6-cyclopentyladenosine (CPA; 200 nM) inhibited spontaneous Ca 2+ oscillations and high potassium (80 mM)-induced Ca 2+ flux in the ventromedial preoptic nucleus, while NEM (100–300 μM) and an A1R antagonist 8-cyclopentyl-dipropylxanthine (300 nM) blocked the CPA actions and increased the high potassium-induced Ca 2+ flux. From these results we suggest that NEM-sensitive G protein-coupled receptor(s) may play an important role in the regulation of sleep and body temperature in the rat and one possible mechanism is an A1R-mediated regulation of intracellular Ca 2+ concentrations in the ventromedial preoptic nucleus.

Thermal denaturation of mixtures of α-lactalbumin and β-lactoglobulin: a differential scanning calorimetric study

The thermal denaturation of α-lactalbumin (α-lac), β-lactoglobulin (β-lg) and a mixture of the two proteins in the presence of several sugars, sodium salts and at various pH values was studied by differential scanning calorimetry. The effects of N-ethylmaleimide (NEM), cysteine, urea and sodium dodecyl sulfate (SDS) were also investigated. The temperature of denaturation ( T d) of β-lg decreased from 71.9°C in the absence of α-lac to 69.1°C in its presence. In contrast, an increase of 2.5°C was observed in the T d of apo-α-lac when heated in the presence of β-lg suggesting that α-lac was made more thermally stable in the presence of β-lg. Glucose and galactose had the greatest effect in stabilizing the proteins against thermal denaturation with the effect being greater for β-lg than for α-lac. A decrease in thermal stability of both proteins was observed in the presence of sodium bicarbonate; sodium ascorbate, however, had a stabilizing effect. Renaturation of α-lac was prevented in the presence of cysteine and NEM, but not in urea or SDS. Translucent gels were formed when the α-lac/β-lg mixtures were heated in the presence of all five sugars and in the presence of cysteine, urea and SDS but not in NEM. This suggests that disulfide–sulfhydryl interchange reactions may be primarily responsible for the gelation of α-lac/β-lg mixtures.

The Effects of Sulfur, Thiol, and Thiol Inhibitor Compounds on Arsine-Induced Toxicity in the Human Erythrocyte Membrane

The mechanism of arsine (AsH(3)) toxicity is not completely understood. The first cytotoxic effect of AsH(3) is disruption of ion homeostasis, with a subsequent hemolytic action. The only accepted treatment for AsH(3) toxicity is exchange transfusion of the blood. In this study the effect of sulfur, sulfur compounds, thiol-containing compounds, and thiol inhibitors on AsH(3)-induced disruption of membrane transport and hemolysis in human erythrocytes was investigated in vitro. Elemental sulfur, sodium thiosulfate, 5, 5'-dithio-bis(2-nitrobenzoic acid), and meso-2,3-dimercaptosuccinic acid were successful in delaying hemolysis, but the most successful agent was the sulfhydryl inhibitor, N-ethylmaleimide (NEM). This indicated that sulfhydryl groups, possibly membrane sulfhydryls, are major factors in the hemolytic mechanism of AsH(3). Measuring intracellular ion concentrations tested the effect of NEM on AsH(3)-induced disruption of membrane transport. AsH(3) alone caused all ions tested to flow with their concentration gradients: Intracellular K+ and Mg++ decreased, whereas Na+, Cl-, and Ca++ increased. NEM was unable to prevent ion loss except for Ca++, whose increase was prevented for 1 h after AsH(3) treatment. The influx of Ca++ in AsH(3)-treated erythrocytes is an irreversible event leading to hemolysis. Reduction of oxygenated hemoglobin to carboxyhemoglobin completely inhibited AsH(3)-induced hemolysis. In addition, AsH(3) and NEM had no direct chemical interactions. We concluded that membrane sulfhydryl groups are likely targets of AsH(3) toxicity, with NEM being able to prevent AsH(3)-induced hemolysis.

The transbilayer distribution of phospholipids in disc membranes is a dynamic equilibrium evidence for rapid flip and flop movement.

We studied the transbilayer redistribution of phospholipids in bovine rod outer segment membranes on thoroughly washed, Ficoll-floated osmotically intact disc vesicles; freshly prepared membranes separated from the disc stack by osmotic shock; and intact disc stacks with a permeabilized plasma membrane (A-discs, B-discs C-discs, respectively). In all cases, spin-labelled phospholipid analogues (SL-PL) with choline, serine and ethanolamine head groups (PtdCho, PtdSer and PtdEtn, respectively) were taken up into the outer leaflet of the membranes by > 90% and within less than 30 s after SL-PL addition, as deduced from the disappearance of spin-label from the suspension medium and from the specific ESR spectrum of membrane-associated spin-label. Using BSA extraction, the amount of SL-PL in the outer leaflet of the bilayer was determined. It decreased with a mean half-time of < 5 min at 25 degrees C, indicating rapid redistribution of all spin-labelled phospholipids into the inner leaflet of the disc membranes. After 1 h, PtdCho and PtdEtn were distributed almost symmetrically, whereas PtdSer was 35 : 65% (in/out). Using subsequent incubation with BSA, the outward movement (flop) of the analogues was observed directly, demonstrating that inward and outward movements proceed in thermodynamic equilibrium. No effect of N-ethylmaleimide or ATP on the redistribution could be measured, which makes it unlikely that energy-consuming translocase or flippase processes are involved in the redistribution in the dark. We reason that the solubilization zone around the photoreceptor rhodopsin may be the locus of rapid redistribution of the highly unsaturated disc phospholipid.