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PubMed2001

Cysteine residues in the D-galactose-H+ symport protein of Escherichia coli: effects of mutagenesis on transport, reaction with N-ethylmaleimide and antibiotic binding.

The galactose-H(+) membrane-transport protein, GalP, of Escherichia coli is similar in substrate specificity and susceptibility to cytochalasin B and forskolin, to the human GLUT1 sugar-transport protein; furthermore, they are about 30% identical in amino acid sequence. Transport activities of both GalP and GLUT1 are inhibited by the thiol-group-specific reagent, N-ethylmaleimide. GalP contains only three cysteine residues at positions 19, 374 and 389, each of which we have mutated, singly and in combination, to serine. Each single change of Cys-->Ser has only a minor effect on transport activity, whereas alteration of all three simultaneously profoundly diminishes V(max) for transport. The high level of expression of the GalP protein facilitates measurements of the reactivity of each mutant with N-ethylmaleimide or eosin 5-maleimide, which conclusively demonstrate that Cys(374) is the site of covalent modification by the reagents. By comparing the reactivity of Cys(374) in right-side-out and inside-out vesicles it appears that Cys(374) is located on the cytoplasmic face of the GalP protein. Although impaired in transport activity, the 'Cys-free' mutant, with all three cysteine residues mutated into serine, binds cytochalasin B and forskolin with wild-type affinities. All these results are interpreted in terms of a 12-helix model of the folding of the protein, in which the relative orientations of helix 10, containing the reactive Cys(374) residue, and helix 11, containing the unreactive Cys(389) residue, can now be defined.

Amino Acid Sequence↗
PubMed1979

A study of the glycerol phosphate acyltransferase and dihydroxyacetone phosphate acyltransferase activities in rat liver mitochondrial and microsomal fractions. Relative distribution in parenchymal and non-parenchymal cells, effects of N-ethylmaleimide, palmitoyl-coenzyme A concentration, starvation, adrenalectomy and anti-insulin serum treatment.

1. GPAT (glycerol phosphate acyltransferase) and DHAPAT (dihydroxyacetone phosphate acyltransferase) activities were measured both in subcellular fractions prepared from fed rat liver and in whole homogenates prepared from freeze-stopped pieces of liver. 2. GPAT activity in mitochondria differed from the microsomal activity in that it was insensitive to N-ethylmaleimide, had a higher affinity towards the palmitoyl-CoA substrate and showed a different response to changes in hormonal and dietary status. 3. Starvation (48 h) significantly decreased mitochondrial GPAT activity. The ratio of mitochondrial to microsomal activities was also significantly decreased. The microsomal activity was unaffected by starvation, except after adrenalectomy, when it was significantly decreased. Mitochondrial GPAT activity was decreased by adrenalectomy in both fed and starved animals. 4. Acute administration of anti-insulin serum significantly decreased mitochondrial GPAT activity after 60 min without affecting the microsomal activity. 5. A new assay is described for DHAPAT. The subcellular distribution of this enzyme differed from that of GPAT. The highest specific activity of DHAPAT was found in a 23 000 gav. pellet obtained by centrifugation of a post-mitochondrial supernatant. This fraction also contained the highest specific activity of the peroxisomal marker uricase. DHAPAT activity in mitochondrial fractions or in the 23 000 gav. pellet was stimulated by N-ethylmaleimide, whereas that in microsomal fractions was slightly inhibited by this reagent. The GPAT and DHAPAT activities in mitochondrial fractions had a considerably higher affinity for the palmitoyl-CoA substrate. 6. Total liver DHAPAT activity was significantly decreased by starvation (48 h), but was unaffected by administration of anti-insulin serum. 7. The specific activities of GPAT and DHAPAT were lower in non-parenchymal cells compared with parenchymal cells, but the GPAT/DHAPAT ratio was 5--6-fold higher in the parenchymal cells.

Acyltransferases↗
PubMed1983

A trypsin-sensitive, heat-labile, N-ethylmaleimide-sensitive factor in adipocyte post-microsomal supernatant which affects the assay of adipocyte glycerol phosphate acyltransferase activities.

Addition of adipocyte 100 000 g post-microsomal supernatant to assays of glycerol phosphate acyltransferase in isolated mitochondria or microsomal fractions decreased activity at lower concentrations of palmitoyl-CoA. At higher concentrations of palmitoyl-CoA, activation was observed on addition of post-microsomal supernatant. The effect of post-microsomal supernatant to decrease activity at lower [palmitoyl-CoA] was abolished by heating or by trypsin treatment, and was also abolished by addition of N-ethylmaleimide to assays or by pretreatment of post-microsomal supernatant with N-ethylmaleimide. The stimulatory effect seen at higher [palmitoyl-CoA] was not sensitive to heat or trypsin treatment. The effect of post-microsomal supernatant at lower [palmitoyl-CoA] cannot be attributed to palmitoyl-CoA hydrolase activity. It was found that brief treatment of adipocyte mitochondria with low concentrations of trypsin was an effective way to remove contaminating microsomal glycerol phosphate acyltransferase activity. Adipocyte post-microsomal supernatant was more effective than an equivalent quantity of liver post-microsomal supernatant protein in decreasing adipocyte microsomal glycerol phosphate acyltransferase activity. The effects of the supernatants from both tissues were decreased by flavaspidic acid. Semi-purified Z-protein fraction from rat liver did not mimic the effect of adipocyte post-microsomal supernatant to decrease glycerol phosphate acyltransferase at lower [palmitoyl-CoA]. Post-microsomal supernatants obtained from noradrenaline-treated adipocytes were less effective than those from control cells in decreasing glycerol phosphate acyltransferase activity in microsomal fractions at lower [palmitoyl-CoA]. It is suggested that adipocyte cytosol may contain an acyl-CoA-binding protein or proteins differing from Z-protein in some respects. The physiological significance of the findings is briefly discussed.

Acyltransferases↗
PubMed1985

Pertussis toxin substrate is a guanosine 5'-[beta-thio]diphosphate-, N-ethylmaleimide-, Mg2+- and temperature-sensitive GTP-binding protein.

We compared the effects of guanine nucleotides and Mg2+ on ADP-ribosylation of rat brain and liver membrane proteins catalysed by Bordetella pertussis toxin (IAP) and cholera toxin (CT). Labelling of proteins in the presence of [alpha-32P]NAD+, ATP and CT required GTP or guanosine 5'-[gamma-thio]triphosphate (GTP [S]). In contrast, labelling of one (liver) or two (brain) polypeptides by IAP was enhanced by guanosine 5'-[beta-thio]diphosphate (GDP[S]) or GTP, but was blocked by GTP[S] or guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG). The order of labelling intensity was GDP[S] greater than GTP greater than no addition greater than GTP[S] = p [NH]ppG. Mg2+ increased labelling by CT, but decreased labelling by IAP. In addition, Mg2+ potentiated the effects of the guanine nucleotides, increasing the inhibitory effects of GTP[S] and the activatory effects of GDP[S] or GTP. Preincubating liver membranes at 30 degrees C in the presence of 10 mm-MgCl2 inhibited labelling by IAP irreversibly. Pretreatment of liver membranes with 4.95 mM-N-ethylmaleimide decreased labelling by CT by approximately 15%, but almost completely blocked labelling by IAP. These results suggest that the undissociated, GDP-bound, conformation of Ni, the inhibitory GTP-binding protein of adenylate cyclase, is the preferred substrate for ADP-ribosylation by IAP. This conformation, which is prevalent in native membranes, is sensitive to temperature, Mg2+ ions and alkylating agents such as N-ethylmaleimide. At 30 degrees C, Mg2+ may cause dissociation and denaturation of Ni in native membranes.

Adenylate Cyclase Toxin↗
PubMed1995

Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver.

N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.

Amines↗
PubMed2003

The nucleotide-binding domains of P-glycoprotein. Functional symmetry in the isolated domain demonstrated by N-ethylmaleimide labelling.

The two nucleotide-binding domains (NBDs) of a number of ATP-binding cassette (ABC) transporters have been shown to be functionally dissimilar, playing different roles in the transport process. A high degree of co-operativity has been determined for the NBDs of the human multidrug transporter, P-glycoprotein. However, the issue of functional symmetry in P-glycoprotein remains contentious. To address this, the NBDs of P-glycoprotein were expressed and purified to 95% homogeneity, as fusions to maltose-binding protein. The NBDs were engineered to contain a single cysteine residue in the Walker-A homology motif. Reactivity of this cysteine residue was demonstrated by specific, time-dependent, covalent labelling with N-ethylmaleimide. No differences in the rates of labelling of the two NBDs were observed. The relative affinity of binding to each NBD was determined for a number of nucleotides by measuring their ability to effect a reduction in N-ethylmaleimide labelling. In general, nucleotides bound identically to the two NBDs, suggesting that there is little asymmetry in the initial step of the transport cycle, namely the recognition and binding of nucleotide. Any observed functional asymmetry in the intact transporter presumably reflects different rates of hydrolysis at the two NBDs or interdomain communications.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Functional and molecular identification of novel members of the ubiquitous membrane fusion proteins alpha- and gamma-SNAP (soluble N-ethylmaleimide-sensitive factor-attachment proteins) families in Dictyostelium discoideum.

The soluble N-ethylmaleimide-sensitive-factor-attachment proteins (SNAP) are eukaryotic soluble proteins required for membrane fusion. Based on their initial identification in bovine brain cytosol, they are divided in alpha/beta and gamma subfamilies. SNAPs act as adapters between N-ethylmaleimide-sensitive factor (NSF), a hexameric ATPase, and membrane SNARE proteins (SNAP receptors). Within the NSF/SNAP/SNARE complex, SNAPs contribute to the catalysis of an ATP-driven conformational change in the SNAREs, resulting in dissociation of the complex. We have constructed a Dictyostelium discoideum strain overexpressing a c-myc-tagged form of D. discoideum NSF (NSF-myc). Its immunoprecipitation from detergent-solubilized membrane extracts reveals two associated polypeptides with apparent molecular masses of 33 and 36 kDa (p33 and p36) that are absent in NSF-myc immunoprecipitates from cytosol. Analysis of trypsin-digested peptides by microsequencing and mass spectrometry and comparison with cDNA sequences identify p33 and p36 as the D. discoideum homologues of alpha- and gamma-SNAP, respectively. The alpha-/gamma-SNAP molar ratio is close to 3 in vegetative amoebae from this organism. The molecular identification of gamma-SNAP in plants (Arabidopsis thaliana) and insects (Drosophila melanogaster) documents, for the first time, the wide distribution of the gamma subtype. Altogether, these results suggest a specific role for gamma-SNAP, distinct from that of alpha-SNAP.

Amino Acid Sequence↗
PubMed2000

Calcium-triggered acrosomal exocytosis in human spermatozoa requires the coordinated activation of Rab3A and N-ethylmaleimide-sensitive factor.

The acrosome reaction of spermatozoa is a complex, calcium-dependent, regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. However, very little is known about the molecules that mediate and regulate this unique fusion process. Here, we show that N-ethylmaleimide-sensitive factor (NSF), a protein essential for most fusion events, is present in the acrosome of several mammalian spermatozoa. Moreover, we demonstrate that calcium-dependent exocytosis of permeabilized sperm requires active NSF. Previously, we have shown that the addition of the active (GTP-bound) form of the small GTPase Rab3A triggers exocytosis in permeabilized spermatozoa. In the present report we show that Rab3A is necessary for calcium-dependent exocytosis. The activation of Rab3A protects NSF from N-ethylmaleimide inhibition and precludes the exchange of the endogenous protein with recombinant dominant negative mutants of NSF. Furthermore, Rab3A activation of acrosomal exocytosis requires active NSF. Our results suggest that, upon calcium stimulation, Rab3A switches to its active GTP-bound form, triggering the formation of a protein complex in which NSF is protected. This process is suggested to be an essential part of the molecular mechanism of membrane fusion leading to the release of the acrosomal contents.

Acrosome↗
PubMed1980

Selective protection of stereospecific enkephalin and opiate binding against inactivation by N-ethylmaleimide: evidence for two classes of opiate receptors.

Stereospecific binding of 3H-labeled [D-Ala2,D-Leu5]enkephalin is irreversibly inactivated by the sulfhydryl group alkylating agent N-ethylmaleimide. This inactivation, like that of opiate binding, exhibits pseudo-first-order kinetics with a half-inactivation time of 10-12 min. The presence of opiates or enkephalins during incubation with N-ethylmaleimide provides good protection. Ouantitative studies of protection demonstrate that naltrexone and morphine are 20 and 8 times, respectively, more effective in protecting the binding of [3H]naltrexone than that of [3H]enkephalin. [D-Ala2,Leu]Enkephalin and [D-Ala2,Met]enkephalin, however, are more effective (7 and 30 times, respectively) for the protection of 3H-labeled [D-Ala2,D-Leu5]enkephalin binding. These results provide strong evidence for the existence of two classes of opiate receptor in rat brain.

Animals↗
PubMed1998

A 29-kilodalton Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptor (Vti1-rp2) implicated in protein trafficking in the secretory pathway.

Expressed sequence tags coding for a potential SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) were revealed during data base searches. The deduced amino acid sequence of the complete coding region predicts a 217-residue protein with a COOH-terminal hydrophobic membrane anchor. Affinity-purified antibodies raised against the cytoplasmic region of this protein specifically detect a 29-kilodalton integral membrane protein enriched in the Golgi membrane. Indirect immunofluorescence microscopy reveals that this protein is mainly associated with the Golgi apparatus. When detergent extracts of the Golgi membrane are incubated with immobilized glutathione S-transferase alpha soluble N-ethylmaleimide-sensitive factor attachment protein (GST-alpha-SNAP), this protein was specifically retained. This protein has been independently identified and termed Vti1-rp2, and it is homologous to Vti1p, a yeast Golgi SNARE. We further show that Vti1-rp2 can be qualitatively coimmunoprecipitated with Golgi syntaxin 5 and syntaxin 6, suggesting that Vti1-rp2 exists in at least two distinct Golgi SNARE complexes. In cells microinjected with antibodies against Vti1-rp2, transport of the envelope protein (G-protein) of vesicular stomatitis virus from the endoplasmic reticulum to the plasma membrane was specifically arrested at the Golgi apparatus, providing further evidence for functional importance of Vti1-rp2 in protein trafficking in the secretory pathway.

Amino Acid Sequence↗
PubMed2004

A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP).

Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor proteins previously proposed to be involved in post-endocytotic sorting of receptors. Of the two proteins suggested to target receptors for recycling to the cell membrane, which is the route believed to be taken by a majority of receptors, ERM (ezrin-radixin-moesin)-binding phosphoprotein 50 (EBP50) bound only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein-coupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the delta-opioid receptor, confirmed the expected nanomolar affinities for interaction with SNX1. Truncations of the NK(1) receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with N-ethylmaleimide-sensitive factor. It is concluded that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrum-recycling adaptor.

Amino Acid Sequence↗
PubMed2004

Insulin and hypertonicity recruit GLUT4 to the plasma membrane of muscle cells by using N-ethylmaleimide-sensitive factor-dependent SNARE mechanisms but different v-SNAREs: role of TI-VAMP.

Insulin and hypertonicity each increase the content of GLUT4 glucose transporters at the surface of muscle cells. Insulin enhances GLUT4 exocytosis without diminishing its endocytosis. The insulin but not the hypertonicity response is reduced by tetanus neurotoxin, which cleaves vesicle-associated membrane protein (VAMP)2 and VAMP3, and is rescued upon introducing tetanus neurotoxin-resistant VAMP2. Here, we show that hypertonicity enhances GLUT4 recycling, compounding its previously shown ability to reduce GLUT4 endocytosis. To examine whether the canonical soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) mechanism is required for the plasma membrane fusion of the tetanus neurotoxin-insensitive GLUT4 vesicles, L6 myoblasts stably expressing myc-tagged GLUT4 (GLUT4myc) were transiently transfected with dominant negative N-ethylmaleimide-sensitive factor (NSF) (DN-NSF) or small-interfering RNA to tetanus neurotoxin-insensitive VAMP (TI-VAMP siRNA). Both strategies markedly reduced the basal level of surface GLUT4myc and the surface gain of GLUT4myc in response to hypertonicity. The insulin effect was abolished by DN-NSF, but only partly reduced by TI-VAMP siRNA. We propose that insulin and hypertonicity recruit GLUT4myc from partly overlapping, but distinct sources defined by VAMP2 and TI-VAMP, respectively.

Animals↗
PubMed1978

The different effects of N-ethylmaleimide and iodoacetamide on the activity of rat liver 60S subunits for peptide bond elongation.

The activity of 60S subunits of rat liver ribosomes in poly(U)-dependent polyphenylalanine synthesis was inhibited by incubation with N-ethylmaleimide. However, when 60S subunits were incubated with iodoacetamide, their activity decreased only slightly. Furthermore, iodoacetamide-pretreated 60S subunits became insensitive to N-ethylmaleimide. Similar results were obtained for the activity of EF-2-dependent GTPase of 60S subunits. As a whole, the labeling patterns of ribosomal proteins on two-dimensional gel electrophoresis were similar for 60S subunits labeled with both 14C-labeled sulfhydryl reagents, although the extent of labeling of some proteins was somewhat different. These results indicate that the SH groups in the 60S subunits are not directly involved in the activities of the subunits described above.

Animals↗
PubMed1982

Inactivation of benzodiazepine binding sites by N-ethylmaleimide.

The benzodiazepine receptors of bovine brain membranes have been identified by the specific binding of radiolabeled [3H]diazepam. Pretreatment of membranes with N-ethylmaleimide causes a dose- and time-dependent decrease of 45 to 60% in the number of binding sites. No decrease occurs when membranes are pretreated with N-ethylmaleimide before administration, or in the presence, of diazepam. Binding of [3H]diazepam to the remaining sites occurs with the same characteristics as binding to the untreated receptor population.

Animals↗
PubMed1991

Partial purification of a novel N-ethylmaleimide-activated translational inhibitor from adult rat brain.

A translational inhibitor that is activated by N-ethylmaleimide treatment can be found in the postmicrosomal fraction prepared from the brain of adult rats, but it is almost undetectable in the same fraction prepared from suckling animals. The inhibitor is thermolabile and remains in the supernatant fraction after precipitation at pH 5. During the purification procedure, the inhibitor in its unactivated state binds to the anion exchanger (diethylaminoethyl-cellulose) but not to the cation exchanger (phosphocellulose). Treatment with N-ethylmaleimide increases inhibitor affinity for the cation exchanger, and this chromatographic step purifies the inhibitor by 143-fold. Both the thermolabile nature and the behavior of the inhibitory activity during the different steps of the purification procedure suggest that this activity is most probably due to a protein. Although the addition of initiation factor 2 reverses the inhibition of protein synthesis in the presence of ATP and Mg2+, the inhibitor does not phosphorylate any of the initiation factor subunits "in vitro," which indicates that it does not contain any intrinsic protein kinase activity. However, its presence in both a crude and a purified preparation of a kinase of the alpha subunit of a brain eukaryotic initiation factor increases the phosphorylation of the alpha subunit of the initiation factor. The mechanism of action of this inhibitor is discussed.

Aging↗
PubMed1992

Prejunctional alpha 2-adrenoceptors in mouse atria function through G-proteins which are sensitive to N-ethylmaleimide, but not pertussis toxin.

1. The identity of the G-proteins involved in prejunctional alpha 2-adrenoceptor signal transduction in mouse atria was examined by use of the G-protein inactivators N-ethylmaleimide and pertussis toxin. 2. The alpha 2-adrenoceptor partial agonist clonidine (0.03 microM) inhibited the electrical stimulation-induced (S-I) outflow of radioactivity from mouse atria which were incubated with [3H]-noradrenaline and stimulated at 5 Hz. The partial alpha 2-adrenoceptor agonist St 363 (10 microM) inhibited the S-I outflow of radioactivity at the lower stimulation frequency of 2.5 Hz. The inhibitory effects of these compounds were not altered in mice pretreated with pertussis toxin (1.5 micrograms, i.v.). 3. The alpha 2-adrenoceptor antagonist, idazoxan (0.1 microM), increased the S-I outflow of radioactivity from mouse atria stimulated at 5 Hz, and this effect was not altered in atria from mice pretreated with pertussis toxin. 4. The inhibitory effects of clonidine and St 363 and the facilitatory effect of idazoxan on the S-I outflow of radioactivity from mouse atria were significantly less in atria incubated with N-ethylmaleimide (NEM, 3 microM) for 60 min before the [3H]-noradrenaline incubation. 5. The results suggest that prejunctional alpha 2-adrenoceptors in mouse atria function through G-proteins which are NEM-sensitive, but pertussis toxin insensitive.

Adrenergic alpha-Agonists↗
PubMed1988

Evidence that prejunctional adenosine receptors regulating acetylcholine release from rat hippocampal slices are linked to an N-ethylmaleimide-sensitive G-protein, but not to adenylate cyclase or dihydropyridine-sensitive Ca2+-channels.

Electrically evoked [3H]acetylcholine ([3H]ACh) release from slices of the rat hippocampus was reduced in a dose-dependent manner by the adenosine A1-receptor agonist R-phenylisopropyladenosine (R-PIA) in the concentration range 0.1-10 microM. The maximal effect was observed with 1 microM R-PIA. Treatment with N-ethylmaleimide (NEM, 100 microM, 10 min), which inactivates nucleotide-binding proteins (G-proteins), caused a slight increase in the basal overflow (0.17 +/- 0.01% v. 0.10 +/- 0.003% in the control slices), but did not affect stimulated release (0.73 +/- 0.05% vs. 0.74 +/- 0.03% in the control slices). N-ethylmaleimide pretreatment significantly reduced the prejunctional inhibitory effect of R-PIA on [3H]ACh release in a non-competitive manner. The S2/S1 ratio was 0.92 +/- 0.03 in controls and was reduced to 0.32 +/- 0.02 by 1 microM R-PIA in the control slices and to 0.57 +/- 0.03 after NEM pretreatment. Stimulation of cyclic AMP-accumulation by forskolin (1 microM) and rolipram (30 microM) before the second stimulation (S2) enhanced the S2/S1 ratio by about 30% to 1.26 +/- 0.12, but did not reduce the inhibitory effect of R-PIA (1 microM). The Ca2+-channel agonist Bay K 86(44) (1 microM), a concentration that increases K+-evoked noradrenaline release, did not affect the basal or electrically evoked [3H]ACh overflow, or the prejunctional effects of R-PIA (0.1 and 1 microM) on [3H]ACh release. Our results suggest that the presynaptic inhibitory effects of A1-receptor agonists on [3H]ACh release are exerted via a nucleotide-binding protein that can be inhibited by NEM.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1997

4 Pro-R hydrogen of NADPH was abstracted for enzymatic hydride transfer by N-ethylmaleimide-reductase of Yarrowia lipolytica.

We studied the steric course of the reaction catalyzed by the N-ethylmaleimide (NEM) reductase of Yarrowia (Candida) lipolytica (Y. lipolytica), using 4R-[4-2H1]NADPH and 4S-[4-2H1]NADPH as cofactors and N-ethylcitraconimide as substrate. Active substrates and inhibitors of NEM reductase and its subcellular distribution were also investigated to clarify the biochemical properties of this enzyme. NEM reductase catalyzes the reduction of N-ethylmaleimide to N-ethylsuccinimide with NAD(P)H as the cofactor. Several maleimide and cyclopentenone derivatives tested were also active substrates for NEM reductase of Y. lipolytica. Some pyrazolone derivatives, particularly 1-phenyl-5-pyrazolone, were found to be effective inhibitors of NEM reductase. Subcellular localization of NEM reductase was carried out using protoplast formation and differential centrifugation. Ninety-eight percent of the NEM reductase activity was recovered in the cytosolic fraction, indicating that NEM reductase in Y. lipolytica was the cytosolic enzyme. We also determined the stereochemical specificity of the reduction of N-ethylcitraconimide by NEM reductase in Y. lipolytica, showing that 4 Pro-R hydrogen of NADPH was abstracted for enzymatic hydride transfer by NEM reductase, and two hydrogen atoms from NADPH and H2O added to opposite faces of the double bond of N-ethylcitraconimide.

Candida↗