N-Ethylmaleimide

Last updated
N-Ethylmaleimide [1]
N-ethylmaleimide.svg
Names
Preferred IUPAC name
1-Ethyl-1H-pyrrole-2,5-dione
Other names
Ethylmaleimide
Identifiers
3D model (JSmol)
AbbreviationsNEM
112448
ChEBI
ChEMBL
ChemSpider
DrugBank
ECHA InfoCard 100.004.449 OOjs UI icon edit-ltr-progressive.svg
EC Number
  • 204-892-4
405614
KEGG
PubChem CID
UNII
  • InChI=1S/C6H7NO2/c1-2-7-5(8)3-4-6(7)9/h3-4H,2H2,1H3 Yes check.svgY
    Key: HDFGOPSGAURCEO-UHFFFAOYSA-N Yes check.svgY
  • InChI=1/C6H7NO2/c1-2-7-5(8)3-4-6(7)9/h3-4H,2H2,1H3
    Key: HDFGOPSGAURCEO-UHFFFAOYAE
  • O=C1\C=C/C(=O)N1CC
Properties
C6H7NO2
Molar mass 125.12528
Melting point 43 to 46 °C (109 to 115 °F; 316 to 319 K)
Boiling point 210 °C (410 °F; 483 K)
Hazards
GHS labelling:
GHS-pictogram-acid.svg GHS-pictogram-skull.svg GHS-pictogram-exclam.svg
Danger
H300, H301, H311, H314, H317
P260, P261, P264, P270, P272, P280, P301+P310, P301+P330+P331, P302+P352, P303+P361+P353, P304+P340, P305+P351+P338, P310, P312, P321, P322, P330, P333+P313, P361, P363, P405, P501
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
X mark.svgN  verify  (what is  Yes check.svgYX mark.svgN ?)

N-Ethylmaleimide (NEM) is an organic compound that is derived from maleic acid. It contains the amide functional group, but more importantly it is an alkene that is reactive toward thiols and is commonly used to modify cysteine residues in proteins and peptides. [2]

Contents

Organic chemistry

NEM is a Michael acceptor in the Michael reaction, which means that it adds nucleophiles such as thiols. The resulting thioether features a strong C–S bond and the reaction is virtually irreversible. Reaction with thiols occur in the pH range 6.5–7.5, NEM may react with amines or undergo hydrolysis at a more alkaline pH. NEM has been widely used to probe the functional role of thiol groups in enzymology. NEM is an irreversible inhibitor of all cysteine peptidases, with alkylation occurring at the active site thiol group (see schematic). [3] [4]

Mechanism of irreversible inhibition of a cysteine peptidase with NEM. NEMmech.jpg
Mechanism of irreversible inhibition of a cysteine peptidase with NEM.

Case studies

NEM blocks vesicular transport. In lysis buffers, 20 to 25  mM of NEM is used to inhibit de-sumoylation of proteins for Western Blot analysis. NEM has also been used as an inhibitor of deubiquitinases.

N-Ethylmaleimide was used by Arthur Kornberg and colleagues to knock out DNA polymerase III in order to compare its activity to that of DNA polymerase I (pol III and I, respectively). Kornberg had been awarded the Nobel Prize for discovering pol I, then believed to be the mechanism of bacterial DNA replication, although in this experiment he showed that pol III was the actual replicative machinery.

NEM activates ouabain-insensitive Cl-dependent K efflux in low-K sheep and goat red blood cells. [5] This discovery contributed to the molecular identification of K–Cl cotransport (KCC) in human embryonic cells transfected by KCC1 isoform cDNA, 16 years later. [6] Since then, NEM has been widely used as a diagnostic tool to uncover or manipulate the membrane presence of K–Cl cotransport in cells of many species in the animal kingdom. [7] Despite repeated unsuccessful attempts to identify chemically the target thiol group, [8] at physiological pH, NEM may form adducts with thiols within protein kinases that phosphorylate KCC at specific serine and threonine residues primarily within the C-terminal domain of the transporter. [9] The ensuing dephosphorylation of KCC by protein phosphatases leads to activation of KCC. [10]

Related Research Articles

<span class="mw-page-title-main">Hypochlorous acid</span> Chemical compound

Hypochlorous acid is an inorganic compound with the chemical formula ClOH, also written as HClO, HOCl, or ClHO. Its structure is H−O−Cl. It is an acid that forms when chlorine dissolves in water, and itself partially dissociates, forming a hypochlorite anion, ClO−. HClO and ClO− are oxidizers, and the primary disinfection agents of chlorine solutions. HClO cannot be isolated from these solutions due to rapid equilibration with its precursor, chlorine.

<span class="mw-page-title-main">Cotransporter</span> Type of membrane transport proteins

Cotransporters are a subcategory of membrane transport proteins (transporters) that couple the favorable movement of one molecule with its concentration gradient and unfavorable movement of another molecule against its concentration gradient. They enable coupled or cotransport and include antiporters and symporters. In general, cotransporters consist of two out of the three classes of integral membrane proteins known as transporters that move molecules and ions across biomembranes. Uniporters are also transporters but move only one type of molecule down its concentration gradient and are not classified as cotransporters.

Sodium-dependent glucose cotransporters are a family of glucose transporter found in the intestinal mucosa (enterocytes) of the small intestine (SGLT1) and the proximal tubule of the nephron. They contribute to renal glucose reabsorption. In the kidneys, 100% of the filtered glucose in the glomerulus has to be reabsorbed along the nephron. If the plasma glucose concentration is too high (hyperglycemia), glucose passes into the urine (glucosuria) because SGLT are saturated with the filtered glucose.

<span class="mw-page-title-main">Na–K–Cl cotransporter</span> Group of transport proteins

The Na–K–Cl cotransporter (NKCC) is a transport protein that aids in the secondary active transport of sodium, potassium, and chloride into cells. In humans there are two isoforms of this membrane transport protein, NKCC1 and NKCC2, encoded by two different genes. Two isoforms of the NKCC1/Slc12a2 gene result from keeping or skipping exon 21 in the final gene product.

<span class="mw-page-title-main">Sodium-chloride symporter</span> Protein-coding gene in the species Homo sapiens

The sodium-chloride symporter (also known as Na+-Cl− cotransporter, NCC or NCCT, or as the thiazide-sensitive Na+-Cl− cotransporter or TSC) is a cotransporter in the kidney which has the function of reabsorbing sodium and chloride ions from the tubular fluid into the cells of the distal convoluted tubule of the nephron. It is a member of the SLC12 cotransporter family of electroneutral cation-coupled chloride cotransporters. In humans, it is encoded by the SLC12A3 gene (solute carrier family 12 member 3) located in 16q13.

In molecular biology, the electroneutral cation-Cl family of proteins are a family of solute carrier proteins. This family includes the products of the Human genes: SLC12A1, SLC12A1, SLC12A2, SLC12A3, SLC12A4, SLC12A5, SLC12A6, SLC12A7, SLC12A8 and SLC12A9.

<span class="mw-page-title-main">Sodium/glucose cotransporter 1</span>

Sodium/glucose cotransporter 1 (SGLT1) also known as solute carrier family 5 member 1 is a protein in humans that is encoded by the SLC5A1 gene which encodes the production of the SGLT1 protein to line the absorptive cells in the small intestine and the epithelial cells of the kidney tubules of the nephron for the purpose of glucose uptake into cells. Recently, it has been seen to have functions that can be considered as promising therapeutic target to treat diabetes and obesity. Through the use of the sodium glucose cotransporter 1 protein, cells are able to obtain glucose which is further utilized to make and store energy for the cell.

<span class="mw-page-title-main">Proteinase K</span> Broad-spectrum serine protease

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<span class="mw-page-title-main">WNK1</span> Protein-coding gene in the species Homo sapiens

WNK , also known as WNK1, is an enzyme that is encoded by the WNK1 gene. WNK1 is serine-threonine protein kinase and part of the "with no lysine/K" kinase WNK family. The predominant role of WNK1 is the regulation of cation-Cl− cotransporters (CCCs) such as the sodium chloride cotransporter (NCC), basolateral Na-K-Cl symporter (NKCC1), and potassium chloride cotransporter (KCC1) located within the kidney. CCCs mediate ion homeostasis and modulate blood pressure by transporting ions in and out of the cell. WNK1 mutations as a result have been implicated in blood pressure disorders/diseases; a prime example being familial hyperkalemic hypertension (FHHt).

<span class="mw-page-title-main">WNK4</span> Protein-coding gene in the species Homo sapiens

Serine/threonine protein kinase WNK4 also known as With No lysine (K) protein kinase 4(WNK4), is an enzyme that in humans is encoded by the WNK4 gene. Missense mutations cause a genetic form of pseudohypoaldosteronism type 2, also called Gordon syndrome or Familial Hyperkalemic Hypertension.

<span class="mw-page-title-main">Chloride potassium symporter 5</span> Protein-coding gene in the species Homo sapiens

Potassium-chloride transporter member 5 is a neuron-specific chloride potassium symporter responsible for establishing the chloride ion gradient in neurons through the maintenance of low intracellular chloride concentrations. It is a critical mediator of synaptic inhibition, cellular protection against excitotoxicity and may also act as a modulator of neuroplasticity. Potassium-chloride transporter member 5 is also known by the names: KCC2 for its ionic substrates, and SLC12A5 for its genetic origin from the SLC12A5 gene in humans.

<span class="mw-page-title-main">Chloride potassium symporter 4</span> Protein-coding gene in the species Homo sapiens

Potassium-chloride transporter, member 4 is a chloride potassium symporter protein. It is encoded by the gene SLC12A4.

<span class="mw-page-title-main">Electroneutral sodium bicarbonate exchanger 1</span> Protein-coding gene in the species Homo sapiens

Electroneutral sodium bicarbonate exchanger 1 is a protein that in humans is encoded by the SLC4A8 gene.

<span class="mw-page-title-main">SLC12A6</span> Protein-coding gene in the species Homo sapiens

Solute carrier family 12 member 6 is a protein that in humans is encoded by the SLC12A6 gene.

<span class="mw-page-title-main">SLC12A7</span> Protein-coding gene in the species Homo sapiens

Solute carrier family 12 member 7 is a protein that in humans is encoded by the SLC12A7 gene.

<span class="mw-page-title-main">STK39</span> Protein-coding gene in the species Homo sapiens

STE20/SPS1-related proline-alanine-rich protein kinase is an enzyme that in humans is encoded by the STK39 gene.

<span class="mw-page-title-main">AATK</span> Protein-coding gene in the species Homo sapiens

Serine/threonine-protein kinase LMTK1 is an enzyme that in humans is encoded by the (AATK) gene.

<span class="mw-page-title-main">Lactose permease</span> Membrane protein

Lactose permease is a membrane protein which is a member of the major facilitator superfamily. Lactose permease can be classified as a symporter, which uses the proton gradient towards the cell to transport β-galactosides such as lactose in the same direction into the cell.

The cation-chloride cotransporter (CCC) family is part of the APC superfamily of secondary carriers. Members of the CCC family are found in animals, plants, fungi and bacteria. Most characterized CCC family proteins are from higher eukaryotes, but one has been partially characterized from Nicotiana tabacum, and homologous ORFs have been sequenced from Caenorhabditis elegans (worm), Saccharomyces cerevisiae (yeast) and Synechococcus sp.. The latter proteins are of unknown function. These proteins show sequence similarity to members of the APC family. CCC family proteins are usually large, and possess 12 putative transmembrane spanners (TMSs) flanked by large N-terminal and C-terminal hydrophilic domains.

The anion exchanger family is a member of the large APC superfamily of secondary carriers. Members of the AE family are generally responsible for the transport of anions across cellular barriers, although their functions may vary. All of them exchange bicarbonate. Characterized protein members of the AE family are found in plants, animals, insects and yeast. Uncharacterized AE homologues may be present in bacteria. Animal AE proteins consist of homodimeric complexes of integral membrane proteins that vary in size from about 900 amino acyl residues to about 1250 residues. Their N-terminal hydrophilic domains may interact with cytoskeletal proteins and therefore play a cell structural role. Some of the currently characterized members of the AE family can be found in the Transporter Classification Database.

References

  1. ↑ N-Ethylmaleimide at Sigma-Aldrich
  2. ↑ "Thiol reactive probes". Archived from the original on 2008-01-28. at Invitrogen
  3. ↑ Nelson, D. L.; Cox, M. M. (2000). Lehninger, Principles of Biochemistry (3rd ed.). New York: Worth Publishing. ISBN   1-57259-153-6.[ page needed ]
  4. ↑ Gregory, J. D. (1955). "The Stability of N-Ethylmaleimide and its Reaction with Sulfhydryl Groups". Journal of the American Chemical Society. 77 (14): 3922–3923. Bibcode:1955JAChS..77.3922G. doi:10.1021/ja01619a073.
  5. ↑ Lauf, P. K.; Theg, B. E. (1980). "A chloride-dependent K+ flux induced by N-ethylmaleimide in genetically low-K+ sheep and goat erythrocytes". Biochemical and Biophysical Research Communications. 92 (4): 1422–1428. doi:10.1016/0006-291X(80)90445-3. PMID   7370044.
  6. ↑ Gillen, C. M.; Brill, S.; Payne, J. A.; Forbush, B., III (1996-07-05). "Molecular cloning and functional expression of the K–Cl cotransporter from rabbit, rat, and human. A new member of the cation-chloride cotransporter family". Journal of Biological Chemistry. 271 (27): 16237–16244. doi: 10.1074/jbc.271.27.16237 . PMID   8663127.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  7. ↑ Adragna, N. C.; Di Fulvio, M.; Lauf, P. K. (2004). "Regulation of K–Cl cotransport: from function to genes". Journal of Membrane Biology. 200: 1–29.
  8. ↑ Lauf, P. K. (1985). "K+ Cl Cotransport: Sulfhydryl, divalent cations and the mechanism of volume activation in a red cell". Journal of Membrane Biology. 88 (1): 1–13. doi:10.1007/BF01871208. PMID   3937898.
  9. ↑ Rinehart, J.; Maksimova, Y. D.; Tanis, J. E.; Stone, K. L.; Hodson, C. A.; Zhang, J.; Risinger, M.; Pan, W.; Wu, D.; Colangelo, C. M.; Forbush, B.; Joiner, C. H.; Gulcicek, E. E.; Gallagher, P. G.; Lifton, R. P. (2009). "Sites of Regulated Phosphorylation that Control K–Cl Cotransporter Activity". Cell. 138 (3): 525–536. doi:10.1016/j.cell.2009.05.031. PMC   2811214 . PMID   19665974.
  10. ↑ Jennings, M. L.; Al-Rohil, N. S. (1990). "Kinetics of activation and inactivation of swelling-stimulated K+/Cl- transport. The volume-sensitive parameter is the rate constant for inactivation". Journal of General Physiology. 95 (6): 1021−1040. doi:10.1085/jgp.95.6.1021. PMC   2216352 . PMID   2373997.
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