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PubMed2001

Partitioning of N-ethylmaleimide-sensitive fusion (NSF) protein function in Drosophila melanogaster: dNSF1 is required in the nervous system, and dNSF2 is required in mesoderm.

The N-ethylmaleimide-sensitive fusion protein (NSF) promotes the fusion of secretory vesicles with target membranes in both regulated and constitutive secretion. While it is thought that a single NSF may perform this function in many eukaryotes, previous work has shown that the Drosophila genome contains two distinct NSF genes, dNSF1 and dNSF2, raising the possibility that each plays a specific secretory role. To explore this possibility, we generated mutations in the dNSF2 gene and used these and novel dNSF1 loss-of-function mutations to analyze the temporal and spatial requirements and the degree of functional redundancy between dNSF1 and dNSF2. Results of this analysis indicate that dNSF1 function is required in the nervous system beginning at the adult stage of development and that dNSF2 function is required in mesoderm beginning at the first instar larval stage of development. Additional evidence suggests that dNSF1 and dNSF2 may play redundant roles during embryonic development and in the larval nervous system. Ectopic expression studies demonstrate that the dNSF1 and dNSF2 gene products can functionally substitute for one another. These results indicate that the Drosophila NSF proteins exhibit similar functional properties, but have evolved distinct tissue-specific roles.

Animals↗

PubMed2002

N-ethylmaleimide-sensitive factor is required to organize functional exocytotic microdomains in paramecium.

In exocytosis, secretory granules contact plasma membrane at sites where microdomains can be observed, which are sometimes marked by intramembranous particle arrays. Such arrays are particularly obvious when membrane fusion is frozen at a subterminal stage, e.g., in neuromuscular junctions and ciliate exocytotic sites. In Paramecium, a genetic approach has shown that the "rosettes" of intramembranous particles are essential for stimulated exocytosis of secretory granules, the trichocysts. The identification of two genes encoding the N-ethylmaleimide-sensitive factor (NSF), a chaperone ATPase involved in organelle docking, prompted us to analyze its potential role in trichocyst exocytosis using a gene-silencing strategy. Here we show that NSF deprivation strongly interferes with rosette assembly but does not disturb the functioning of exocytotic sites already formed. We conclude that rosette organization involves ubiquitous partners of the fusion machinery and discuss where NSF could intervene in this mechanism.

Animals↗

PubMed1987

Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. I. Location of a group which is most reactive with N-ethylmaleimide.

Ca2+-Transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum contains several SH groups which are reactive with N-ethylmaleimide (MalNEt) at pH 7.0. The location of the one which is most reactive with MalNEt (SHN, Kawakita et al. J. Biochem. 87, 609 (1980)) was identified on the amino acid sequence of the ATPase. SHN was labeled by reacting sarcoplasmic reticulum membranes with [14C] MalNEt to a labeling density of 1 mol/mol ATPase. [14C]MalNEt-labeled membranes were digested with thermolysin and 14C-labeled SHN peptides were fractionated by Sephadex LH-20 chromatography to give two major peaks of radioactivity. [14C]-MalNEt-labeled peptides were further purified to homogeneity by C18-reversed phase HPLC. Two radioactive peptides containing modified cysteine (Cys), Leu-Gly-Cys-Thr-Ser and Val-Cys-Lys-Met, were finally obtained in roughly equal amounts and in reasonable recovery. Both of these sequences were found in the amino acid sequence of Ca2+-transporting ATPase (Brandl et al. Cell 44, 597 (1986)), and Cys344 and Cys364 were identified as the targets of MalNEt-modification. Thus, 0.5 mol/mol ATPase of each Cys residue actually reacted rapidly with MalNEt under the conditions leading to SHN-modification. Modification of either one with MalNEt may negatively affect the reactivity of the other. Both of the highly reactive SH groups are located in the neighborhood of Asp351, the phosphorylation site of ATPase.

Adenosine Triphosphatases↗

PubMed1987

Effect of N-ethylmaleimide on Ca-inhibition of Physarum myosin.

We have established a quick method for preparing Physarum myosins whose actin-activated ATPase activities are inhibited by microM levels of Ca2+ (from plasmodial stage: Kohama, K. & Kendrick-Jones, J. (1986) J. Biochem. 99, 1433-1446; and from amoebal stage: Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, Y., & Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). N-Ethylmaleimide alkylates sulfhydryl (SH) groups on the heavy chains in the heads of the plasmodial myosin. The actin-activated ATPase activity of the modified myosin was significantly decreased when assayed in low Ca2+ concentrations. Moreover, the activity remained low even when the Ca2+ concentrations was increased, i.e., the myosin was desensitized. For complete desensitization, about 4 mol SH per mol myosin (500,000 Mr) must be modified. These residues are probably the "reactive thiols" which have been predicted from primary structure studies to be conserved among myosins of higher and lower eukaryotes. Ultraviolet absorption spectra of the modified and intact myosins showed a peak at 277 nm. The height of this peak in intact myosin was reduced when the Ca2+ concentration was increased. This Ca-induced reduction was hardly detectable in the modified myosin although Ca-binding activity to myosin did not appear to be affected by the modification. We interprete these results that Ca2+ may change the conformation of the myosin heavy chain by binding to myosin and speculate that impairment of this process upon modification could cause the desensitization to Ca2+ in the ATPase activity.

Actins↗

PubMed1989

Purification and characterization of N-ethylmaleimide reducing enzyme from Candida lipolytica.

N-Ethylmaleimide (NEM) reducing enzyme was purified to homogeneity from cell-free extracts of Candida lipolytica by chromatography techniques. The molecular weight of the native enzyme was estimated to be about 43,000 by gel filtration using Superose 12 and to be 47,000 by SDS-PAGE. This enzyme can use both NADPH and NADH as an electron donor, and catalyzes the reduction of the carbon-carbon double bond of five membered ring compounds which have two conjugated carbonyl groups on both sides of a double bond.

Amino Acids↗

PubMed1990

Determination of the primary structure of intermolecular cross-linking sites on the Ca2(+)-ATPase of sarcoplasmic reticulum using 14C-labeled N,N'-(1,4-phenylene)bismaleimide or N-ethylmaleimide.

To determine the intermolecular cross-linking site on the primary structure sarcoplasmic reticulum (SR) Ca-ATPase, the conditions for the specific binding of 14C-labeled 1,4-phenylene bis maleimide (PBM) or 14C-labeled N-ethylmaleimide (NEM) to the ATPase were explored. SR vesicles were preincubated with nonradioactive PBM in the presence of 1 mM vanadate for 1 h, then washed by centrifugation to remove free PBM and vanadate. When the pretreated SR vesicles were allowed to react with 1 mM [14C]PBM in the presence of 1 mM AMPPNP, the amount of [14C]PBM incorporated into the ATPase increased with time in parallel with the formation of dimeric ATPase and reached the maximum labeling density of 1 mol of [14C]PBM per mol of dimeric ATPase at 40 min after the start of the reaction. When the pretreated SR vesicles were allowed to react with 2 mM [14C]NEM in the absence of AMPPNP, a maximum of about 2 mol of NEM was bound per mol of the ATPase monomer. The labeling density of [14C]NEM decreased from 2 to 1 mol per mol of the ATPase when the SR vesicles were allowed to react with [14C]NEM in the presence of AMPPNP. From the analysis of the amino acid composition of the two major [14C]NEM-labeled peptides isolated from the thermolytic digest of the enzyme after the reaction of SR with [14C]NEM in the absence of AMPPNP, we deduced that [14C]NEM was incorporated into Cys377 and Cys614. On the other hand, the labeling of SR in the presence of AMPPNP resulted in inhibition of the [14C]NEM binding to Cys614, leaving Cys377 unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1976

Chemical modification of the Ca2+-dependent ATPase of sarcoplasmic reticulum from skeletal muscle. I. Binding of N-ethylmaleimide to sarcoplasmic reticulum: evidence for sulfhydryl groups in the active site of ATPase and for conformational changes induced by adenosine tri- and diphosphate.

The time course of binding of N-ethylmaleimide (NEM) to the SR was measured at pH 7.5 in the presence or absence of ATP or ADP. The following results were obtained. 1. Both in the presence and absence of nucleotide, the ATPase [EC 3.6.1.3] activity decreased linearly with increase in the amount of NEM bound to the fragmented sarcoplasmic reticulum (SR), and was inhibited almost completely by the binding of 2 moles of NEM per 10(5) g of the SR protein. 2. The amount of NEM incorporated into the ATPase (M.W.=105,000) was measured by SDS disc-gel electrophoresis. It was shown that the ATPase activity was inhibited almost completely by the binding of 2 moles of NEM per mole of ATPase. 3. The rate of binding of NEM to SR decreased by 30-40% in the presence of either ATP or ADP. The concentrations of both ATP and ADP for half-saturation were 0.1-0.2mM. 4. The effect of nucleotide on the rate of binding of NEM was not changed by the presence of Ca2+ and Mg2+ ions. Similar effects were also observed even when the SR membranes were solubilized with Triton X-100. It is suggested from these results that one or two SH groups are located in the active site of the SR ATPase, and that conformational changes are induced by the addition of ATP and ADP.

Adenosine Diphosphate↗

PubMed1977

Effects of adenosine triphosphate on N-ethylmaleimide-induced modification of 30S dynein from Tetrahymena cilia.

Ciliary 30S dynein of Tetrahymena was investigated with regard to modification of the ATPase activity with N-ethylmaleimide (NEM) in the presence of ATP. The elevation of enzyme activity due to the modification was largely repressed by addition of ATP at a concentration of 1 mM or more during preincubation of 20 h at 0 degrees C. The repression was highly specific for ATP, though ADP and AMPPNP showed slight repressive effects. After complete hydrolysis of ATP added to the preincubation mixture, however, elevation of 30S dynein ATPase activity occurred. It is suggested that the repression by ATP of NEM-induced elevation of 30S dynein ATPase activity is simply due to a protecting effect of ATP on certain SH group(s) (probably SH1-type group(s)) around the active center of 30S dynein. When 30S dynein was maximally activated by modification with NEM, ATP or ADP did not significantly promote the inactivation of the modified enzyme upon further treatment with NEM, indicating that 30S dynein lacks the characteristics of SH2-type groups. On the other hand, ATP also showed a protective effect against inhibition of native 30S dynein by high concentrations of NEM. High concentrations of ADP and AMPPNP were inhibitory to 30S dynein ATPase activity but inorganic phosphate did not inhibit 14S or 30S dynein ATPase activities at all.

Adenosine Diphosphate↗

PubMed1980

Selective modification of functionally distinct sulfhydryl groups of sarcoplasmic reticulum Ca2+,Mg2+-adenosine triphosphatase with N-ethylmaleimide.

Rabbit sarcoplasmic reticulum vesicles were treated with N-ethylmaleimide (NEM) at pH 7.0. At 1.5 mM NEM, only 4 SH groups per mol of ATPase peptide were modified in 25 min at 30 degrees C. Two of these are essential for Ca2+ transport, one being involved in E-P formation (SHF), and the other in its decomposition (SHD), whereas the other two are apparently non-essential (SHN and SHN'). SHN was modified first, followed by SHD, SHN', and SHF, in this order. Modification of SHD was accompanied by the loss of Ca2+-transport activity, while E-P forming activity survived until the least reactive one (SHF) was modified. At a lower NEM concentration (4 x 10(-5) M) SHN could be selectively modified without loss of the enzyme activity. SHF could be protected by adenyl-5'-yl-imidodiphosphate (AMP-P(NH)P) in the presence of Ca2+ ions, whereas SHD was not. SHD was distinctly less reactive in the absence of Ca2+ (less than 10(-7) M) than in its presence. Changes in the reactivity of these SH groups may be related to conformational changes of the ATPase molecule induced by the binding of Ca2+ and ATP.

Adenylyl Imidodiphosphate↗

PubMed1981

The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. VII. A chemical modification of colonic smooth myosin B with N-ethylmaleimide.

The reactivity of the sulfhydryl groups in colonic myosin B to N-ethylmaleimide (NEM) was studied under various conditions. A higher concentration of NEM was required to alter the ATPase activity as compared with skeletal myosin B. A chemical modification of colonic myosin B at a low ionic strength brought about the marked inhibition of EDTA-ATPase and 30-40% activation of Mg2+-ATPase, but had little effect on Ca2+-ATPase. The same results were obtained with NEM modification at a high ionic strength. The presence of 10 mM MgCl2 in the reaction medium for NEM modification caused the activation of Ca2+-ATPase and remarkable inhibition of EDTA-ATPase, suggesting that SH1 is here reactive to NEM. NEM modification of colonic myosin B in the presence of ATP resulted in the inhibition of not only EDTA-ATPase but also Ca2+- and Mg2+-ATPases although the degree of inhibition was different. Addition of 2.0 M urea to the modification medium caused the simultaneous inhibition of EDTA- and Ca2+-ATPase but little change in Mg2+-ATPase. Based on these results, the presence of three specific sulfhydryl groups, SHa, SH1, and SH2, is discussed.

Adenosine Triphosphatases↗

PubMed1982

Effect of concentration of potassium salts on the activity of the N-ethylmaleimide-treated hemin-controlled translational inhibitor.

The effect of various concentrations of potassium salts on the activity of the N-ethylmaleimide(NEM)-treated hemin-controlled translational inhibitor (HCI) was investigated using the rabbit reticulocyte lysate system. The NEM-treated HCI was found to be less active for the inhibition of globin synthesis at higher concentrations of potassium salts. In addition, the binding of [35S]Met-tRNAf to the 40S ribosomal subunits was inhibited more profoundly at 100 mM than at 240 mM KOAc. The phosphorylation of the NEM-treated HCI and eukaryotic initiation factor, eIF-2, was studied at various concentrations of KOAc. The NEM-treated HCI was phosphorylated to an almost constant extent at 20 to 300 mM K+, but the phosphorylation of eIF-2 alpha by the inhibitor decreased with increasing concentration of KOAc. It was also found that the incubation of the 32P-labeled inhibitor with eIF-2 did not produce the 32P-labeled eIF-2. In the light of these data, a possible relationship between the phosphorylation of the inhibitor or eIF-2 alpha and the inhibition of globin synthesis by the inhibitor is discussed.

Animals↗

PubMed1985

Specific phosphorylation of pig liver initiation factor eIF-2 by the N-ethylmaleimide-treated hemin-controlled translational inhibitor.

The specific phosphorylation of pig liver initiation factor 2(eIF-2) by the N-ethylmaleimide (NEM)-treated hemin-controlled translational inhibitor (HCI) from rabbit reticulocytes was investigated. The inhibitor phosphorylated the serine residue of the alpha subunit of eIF-2 (eIF-2 alpha) and 1 mol of phosphate was incorporated into 1 mol of eIF-2 alpha by the inhibitor on maximal phosphorylation, even when eIF-2 was pretreated with alkaline phosphatase prior to phosphorylation. The 32P-labeled eIF-2 alpha was subjected to tryptic digestion and the tryptic digest was analyzed by two-dimensional peptide mapping on a cellulose thin-layer sheet. After 94 h digestion, the autoradiograph of the peptide map showed a single 32P-labeled band with a molecular weight of approximately 1,200. These findings suggest that one specific serine residue of pig liver eIF-2 alpha was phosphorylated by the NEM-treated HCI.

Animals↗

PubMed1995

Localization of N-ethylmaleimide-sensitive fusion protein in pinealocytes.

N-ethylmaleimide-sensitive fusion protein (NSF), a protein necessary for vesicular docking and/or fusion, was detected immunohistochemically in pinealocytes. NSF was distributed similarly to synaptophysin and vacuolar-type H(+)-ATPase (V-ATPase), marker proteins for synaptic-like microvesicles (MVs) abundantly present in pinealocytes. A subcellular fractionation study indicated that .> 95% of NSF was present as a membrane-bound form and that some NSF was associated with MVs. Like neuronal NSF, the protein was not solubilized from membranes with either 2 mM Mg-ATP or 2% sodium carbonate, suggesting that NSF was tightly bound to the membranes. NSF was also detected in purified MVs from bovine posterior pituitaries. Since MVs are the organelles in which transmitters are stored, these results suggest that NSF is involved in the MV-mediated exocytosis of transmitters from endocrine cells.

Adrenal Medulla↗

PubMedDate not supplied

Role of sulfhydryl groups in aortic responses using N-ethylmaleimide.

This study examines the effects of N-ethylmaleimide (NEM) 10(-7) M on agonist-induced contraction, and the relaxation following drug washout of vascular smooth muscle (VSM) segments. 45Ca flux, cyclic 3':5'-adenosine-monophosphate (cAMP), and cyclic 3':5'-guanosine-monophosphate (cGMP) were analyzed in an attempt to understand the observed physiological modifications with NEM. Cyclic nucleotides were measured by 125I-radioimmunoassay. Contractile responses to norepinephrine (NE) 5.9 x 10(-7) M, angiotensin II (AT) 9.8 x 10(-8) M, and potassium chloride (KCl) 2.2 x 10(-2) M were significantly depressed when VSM segments were exposed to NEM prior to agonist challenge. An inhibition of relaxation occurred when NEM was added after the development of a maximal contractile response to NE, AT, or KCl. VSM relaxation was significantly inhibited when compared to controls. Altered 45Ca efflux, increased cGMP, and decreased cAMP levels were all associated with an inhibition of relaxation of VSM. These results suggest a regulatory role of sulfhydryl (SH) groups in contraction and relaxation of VSM involving both the control of Ca2+ flux and cAMP and cGMP metabolism.

Animals↗

PubMed1992

N-ethylmaleimide diminishes alpha 2-adrenoceptor-mediated effects on norepinephrine release in rat tail arteries.

Isolated tail arteries from Wistar rats, prelabeled with [3H]norepinephrine (NE) were subjected to electrical field stimulation (24 pulses at 0.4 Hz and 200 mA). Both NE release and vasoconstriction were measured in parallel. The selective alpha 2-adrenoceptor agonist B-HT 933 diminished the evoked NE release in a concentration-dependent manner. This effect of B-HT 933 was counteracted by the selective alpha 2-adrenoceptor antagonist rauwolscine, which given alone enhanced evoked transmitter release, indicating the presence of autoinhibition. N-Ethylmaleimide (NEM) (3 microM), which also in itself increased transmitter release, virtually abolished facilitation of release by 0.1 microM rauwolscine and diminished its inhibition by 10 microM B-HT 933. The diminution of the inhibitory effect of B-HT 933 was even more pronounced when the current strength was decreased from 200 mA to 90 mA to compensate for the NEM-induced increase in transmitter release. Treatment of the arteries with NEM did not affect the perfusion pressure. In contrast, however, the B-HT 933-induced increase in basal perfusion pressure was significantly diminished by NEM. Although 10 microM B-HT 933 given alone did not affect stimulation-evoked vasoconstriction, it caused a significant increase in arteries treated with NEM. In conclusion, the observed NEM-sensitivity of the presynaptic and vascular alpha 2-adrenoceptor mechanisms is compatible with the idea that both pre- and postsynaptic alpha 2-adrenoceptors couple to Pertussis toxin (PTX)-sensitive G proteins.

Animals↗

PubMed1985

Beta-endorphin does not protect alkylation of opiate receptor by N-ethylmaleimide.

Rat brain membranes were incubated in N-ethylmaleimide (NEM, 0.5-1.0 mM) in the presence and absence of various concentrations of morphine, Leuenkephalin and human beta-endorphin (beta h-EP). After sufficient washing, the binding of dihydromorphine (DHM), [D-Ala-D-Leu]-enkephalin (DADLE) and tritiated beta h-EP was 10-40% above that of membranes treated with NEM alone. There was no additive effect of morphine and Leu-enkephalin with respect to their effect on recovery of beta h-EP binding. Evaluation of beta h-EP as protecting ligand proved to be difficult since preincubation completely inhibits subsequent DHM and DADLE binding unless a more extensive washing protocol is employed. A protocol for washing beta h-EP preincubated membranes using a Tris-phosphate buffer of pH 6 containing 150 mM NaCl, 20 mM MgCl2 and 10% glycerol was used to recover enough binding potential to evaluate the effects of beta h-EP preincubation towards NEM treatment. Preincubation with beta h-EP itself at 0.1-1.0 microM did not result in any increased recovery of opiate binding, in contrast to the findings with the other two ligands.

Alkylation↗

PubMed1979

An investigation of the conformational properties of ribosomes using N-ethylmaleimide as a probe.

The reactivity of ribosomal proteins towards N-ethylmaleimide has been examined in a variety of ribosome and ribosomal subunit preparations from Escherichia coli. The data show that samples which would be regarded as equivalent operationally can differ significantly in conformation, as judged by reactivity, depending on the method of preparation. The washing of ribosomes with high concentrations of salt has a particularly dramatic effect on protein reactivity. The implications of these results for our understanding of ribosome conformation and for the further study of conformation by chemical reactivity are discussed.

Centrifugation, Density Gradient↗

PubMed2006

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.

Animals↗