Ethylmaleimide

EC 3.4.24.3]. Purified by using /V-ethylmaleimide to activate the enzyme, and wheat germ agglutinin-agarose affinity chromatography [Callaway et al. Biochemistry 25 4757 1986],... 

In recent years, biochemists have developed an arsenal of reactions that are relatively specific to the side chains of particular amino acids. These reactions can be used to identify functional amino acids at the active sites of enzymes or to label proteins with appropriate reagents for further study. Cysteine residues in proteins, for example, react with one another to form disulfide species and also react with a number of reagents, including maleimides (typically A ethylmaleimide), as shown in Figure 4.11. Cysteines also react effectively... 

Aqueous solutions of aequorin also emit light upon the addition of various thiol-modification reagents, such as p-quinone, Br2, I2, N-bromosuccinimide, N-ethylmaleimide, iodoacetic acid, and p-hydroxymercuribenzoate (Shimomura et al., 1974b). The luminescence is weak and long-lasting ( 1 hour). The quantum yield varies with the conditions, but seldom exceeds 0.02 at 23-25°C. The luminescence is presumably due to destabilization of the functional moiety caused by the modification of thiol and other groups on the aequorin molecule. 

Mild chromic acid oxidation of luciferin (CrOs/KHSC /HiO, room temperature) yielded 3-methyl-4-vinylmaleimide (1, Fig. 8.7), 3-methyl-4-ethylmaleimide (2), and an aldehyde (3), whereas vigorous chromic acid oxidation (CrOs/2N H2SO4, 90°C) gave hema-tinic acid (4) (Dunlap et al., 1981). These results closely resemble the results of the chromic acid oxidation of the fluorescent compound F of euphausiid (p. 76), indicating a structural similarity between dinoflagellate luciferin and the compound F. 

Lipid phosphate phosphohydrolases (LPPs), formerly called type 2 phosphatidate phosphohydrolases (PAP-2), catalyse the dephosphorylation of bioactive phospholipids (phosphatidic acid, ceramide-1-phosphate) and lysophospholipids (lysophosphatidic acid, sphingosine-1-phosphate). The substrate selectivity of individual LPPs is broad in contrast to the related sphingosine-1-phosphate phosphatase. LPPs are characterized by a lack of requirement for Mg2+ and insensitivity to N-ethylmaleimide. Three subtypes (LPP-1, LPP-2, LPP-3) have been identified in mammals. These enzymes have six putative transmembrane domains and three highly conserved domains that are characteristic of a phosphatase superfamily. Whether LPPs cleave extracellular mediators or rather have an influence on intracellular lipid phosphate concentrations is still a matter of debate. 

Catalytic antibody 1E9, the first catalytic antibody discovered for Diels-Alder reaction, catalyzes the cycloaddition between tetrachlorothiophene dioxide and N-ethylmaleimide (Equation 4.15) [86]. 

Rideout and Breslow first reported [2a] the kinetic data for the accelerating effect of water, for the Diels Alder reactions of cyclopentadiene with methyl vinyl ketone and acrylonitrile and the cycloaddition of anthracene-9-carbinol with N-ethylmaleimide, giving impetus to research in this area (Table 6.1). The reaction in water is 28 to 740 times faster than in the apolar hydrocarbon isooctane. By adding lithium chloride (salting-out agent) the reaction rate increases 2.5 times further, while the presence of guanidinium chloride decreases it. The authors suggested that this exceptional effect of water is the result of a combination of two factors the polarity of the medium and the... 

Table 6.2 Sodium and guanidinium salt effects (relative reaction rates) of Diels-Alder reaction of anthracene-9-carbinol and N-ethylmaleimide...

NEM N-Ethylmaleimide, a chemical that alkylates sulfhy-dryl groups... 

Step 7 Hydrolysis of ATP by NSF is essential for fusion, a process that can be inhibited by NEM N-ethylmaleimide). Gertain other proteins and calcium are also required. 

Of the 20 residues that react with A-ethylmaleimide in the non-reduced denatured Ca -ATPase at least 15 are available for reaction with various SH reagents in the native enzyme [75,239,310]. These residues are all exposed on the cytoplasmic surface. After reaction of these SH groups with Hg-phenyl azoferritin, tightly packed ferritin particles can be seen by electron microscopy only on the outer surface of the sarcoplasmic reticulum vesicles [143,311-314]. Even after the vesicles were ruptured by sonication, aging, or exposure to distilled water, alkaline solutions or oleate, the asymmetric localization of the ferritin particles on the outer surface was preserved [311,313,314]. 

Identification of cysteine residues that react with N-ethylmaleimide (MalNEt)... 

Chemical modifications like alkylation with (A-ethylmaleimide (NEM) or oxidation with diamide that inhibit the phosphorylation activity of the enzyme did not seem to have any significant effect on the high affinity binding site when the enzyme was solubilized in the detergent decyl-PEG [69,41]. However, in the intact membrane these treatments reduced the affinity by a factor of 2-3. The reduction of the affinity was exclusively due to modification of the cysteine residue at position 384 in the B domain [69]. Apparently, the detergent effects the interaction between the B and C domains. 

The history of observations of efflux associated with PTS carriers is nearly as old as PTS itself. Gachelin [82] reported that A -ethylmaleimide inactivation of a-methyl-glucoside transport and phosphorylation in E. coli was accompanied by the appearance of a facilitated diffusion movement of both a-methylglucoside and glucose in both directions, uptake and efflux. His results could not discriminate, however, between one carrier operating in two different modes, active transport for the native carrier and facilitated diffusion for the alkylated carrier, or two distinct carriers. Haguenauer and Kepes [83] went on to show that alkylation of the carrier was not even necessary to achieve efflux NaF treatment which inhibits P-enolpyruvate synthesis was sufficient but this study did not address the question of one carrier or two. 

Abbreviations DEPC, diethylpyrocarbonate DCCD, Af.iV -dicyclohexylcarbodiimide EEDQ, N-ethyoxycarbonyl-2-ethoxy-1,2-dihydroquinoline NEM, A-ethylmaleimide PAO, phenylarsine oxide NPM, IV-phenylmaleimide PLP, pyridoxal phosphate DIDS, diisothiocyanostilbene disulfonate PITC, phenylisothiocyanate. 

Miura K, Y Tomioka, H Suzuki, M Yonezawa, T Hishinuma, M Mizugaki (1997) Molecular cloning of the nemA gene encoding A -ethylmaleimide reductase from Escherichia coli. Biol Pharm Bull 20 110-112. 

On the other hand, the use of a-cyclodextrin decreased the rate of the reaction. This inhibition was explained by the fact that the relatively smaller cavity can only accommodate the binding of cyclopentadiene, leaving no room for the dienophile. Similar results were observed between the reaction of cyclopentadiene and acrylonitrile. The reaction between hydroxymethylanthracene and N-ethylmaleimide in water at 45°C has a second-order rate constant over 200 times larger than in acetonitrile (Eq. 12.2). In this case, the P-cyclodextrin became an inhibitor rather than an activator due to the even larger transition state, which cannot fit into its cavity. A slight deactivation was also observed with a salting-in salt solution (e.g., quanidinium chloride aqueous solution). 

Optically active Diels-Alder adducts were also prepared by using a one-pot preparative method and enantioselective formation of inclusion complex with optically active hosts in a water suspension medium.68 For example, A-ethylmaleimide reacts with 2-methyl-1,3-butadiene in water to give the racemic adduct 1. Racemic 1 and the optically active host 2 form enantioselectively a 1 1 inclusion complex of 2 with (+)-l in a water suspension. The inclusion complex can be filtered and heated to release (+)-l with 94% ee (Eq. 12.23). 

ND = Not determined DTNB 2,2 -Dithiobis(5-nitropyridine) DTNP 2,2 -Dithiobis(5-nitrobenzoate) DEDC Diethyldithiocarbamate IA Iodoacetate NEM N-ethylmaleimide PCMB p-chloromercuribenzoate 1, 10-PT 1, 10-phenanthroline 8-Q 8-Quinolinol 2, 2 -BP 2, 2 -Bipyridyl. 

Gorin, G., Martin, P.A., and Doughty, G. (1966) Kinetics of the reaction of N-ethylmaleimide with cysteine and some congeners. Arch. Biochem. Biophys. 115, 593. 

Haugaard, N., Cutler, J., and Ruggieri, M.R. (1981) Use of N-ethylmaleimide to prevent interference by sulfhydryl reagents with the glucose oxidase assay for glucose. Anal. Biochem. 116, 341-343. 

Smyth, D.G. (1967) Acetylation of amino and tyrosine hydroxyl groups./. Biol. Chem. 242, 1592-1598. Smyth, D.G., Blumenfeld, O.O., and Konigsberg, W. (1964) Reaction of N-ethylmaleimide with peptides and amino acids. Biochem. J. 91, 589. 

N-ethylmaleimide-sensitive Trimeric ATPase required for in vitro membrane fusion during vesicular transport. Probably function as factor (NSF) chaperones in synaptic vesicle recycling. 

NSF N-ethylmaleimide-sensitive factor PNUTS phosphatase 1 nuclear targeting subunit... 

Cysteine mutations in GAT verified an intracellular position for Cys3998.63 in the fourth intracellular loop (IL4) between TM8 and TM9 (17), and an extracellular location for positions 73i.55-76i.58 in ELI (15), as cysteines at these positions conferred sensitivity to the sulfhydryl reagents, MTSEA and A-ethylmaleimide (for Cys39 98.63) and MTSET (residues 73i.55-76i.58). 

It has recently been shown that the rates of certain Diels-Alder reactions are also accelerated dramatically when carried out in perfluorinated solvents [10]. When the reaction between A -ethylmaleimide and 9-hydroxymethylanthracene (Scheme 7.4) was examined in a range of solvents, significant rate enhancements were found in water and perfluorinated solvents compared to organic solvents. 

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