Met5 enkephalin

Manufactured by Merck Group

[Met5] enkephalin is a pentapeptide that functions as an endogenous opioid peptide. It consists of five amino acids and is involved in pain relief and modulation of the nervous system.

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4 protocols using met5 enkephalin

Pharmacological Manipulation of Opioid Signaling

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Morphine sulfate and morphine alkaloid were obtained from the National Institute on Drug Abuse (NIDA), Neuroscience Center (Bethesda, MD). Naloxone was purchased from Abcam (Cambridge, MA), MK-801, from Hello Bio (Princeton, NJ), UK14304 tartrate and idazoxan (Ida) from Tocris (Bio-Techne Corp. Minneapolis, MN). Potassium methanesulfonate was from Alfa Aesar (Ward Hill, MA). [Met⁵] enkephalin (ME), endomorphin-1, muscarine, scopolamine, idazoxan and other reagents were from Sigma (St. Louis, MO). Caged-enkephalin (CYLE) and Caged-Naloxone (CNV-Nal) were gifts from Mathew Banghart.
Morphine alkaloid was converted to salt form with 0.1 M HCl and made up a stock solution in water. The working solution was diluted in artificial cerebrospinal fluid (ACSF) and applied during incubation or superfusion. Naloxone, endomorphin-1, muscarine, scopolamine, UK14304 tartrate and idazoxan were dissolved in water, diluted in ACSF and applied by bath superfusion. Bath perfusion of ME was with bestatin (10 mM) and thiorphan (1 mM) to limit breakdown of ME.

Arttamangkul S., Heinz D.A., Bunzow J.R., Song X, & Williams J.T. (2018). Cellular tolerance at the µ-opioid receptor is phosphorylation dependent. eLife, 7, e34989.

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Peptide Characterization by Mass Spectrometry

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Water, methanol, and trifluoroacetic acid (TFA) used as solvents and additives throughout these experiments were obtained from Fisher Scientific (Optima LC/MS grade). The modified peptide renin substrate I (97%), human angiotensin I (90%), human angiotensin II (93%), bradykinin (98%), substance P (95%), Met⁵-enkephalin (95%), and [d-Ala²,d-Leu⁵]-enkephalin (95%) were all purchased as acetate salts from Sigma-Aldrich (St. Louis, MO). Biotinylated, phosphorylated SAMS peptide was obtained from Enzo Life Sciences (Farmingdale, NY). Support solutions for the SCGD were prepared by dilution of concentrated, trace metal grade hydrochloric acid (J.T. Baker, Center Valley, PA) with deionized water of 18.2-MΩ resistivity, prepared in-house with a mixed-bed ion-exchange deionization unit.

Schwartz A.J., Shelley J.T., Walton C.L., Williams K.L, & Hieftje G.M. (2016). Atmospheric-pressure ionization and fragmentation of peptides by solution-cathode glow discharge †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02032a Click here for additional data file.. Chemical Science, 7(10), 6440-6449.

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Electrophysiological Opioid Receptor Assay

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Naloxone and MK-801 were purchased from Hello Bio (Princeton, NJ). [Met⁵]enkephalin and CTAP were purchased from Sigma (St. Louis, MO). UK14304 tartrate and idazoxan were from Tocris (R and D system, Minneapolis, MN). Morphine alkaloid was obtained from National Institute on Drug Abuse, Neuroscience Center (Bethesda, MD) and was converted to HCl salt with 0.1 M HCl as a stock solution. All drugs were diluted to the tested concentrations in artificial cerebrospinal fluid (ACSF) and applied during superfusion. All salts used in electrophysiological experiments were purchased from Sigma.

Arttamangkul S., Plazek A., Platt E.J., Jin H., Murray T.F., Birdsong W.T., Rice K.C., Farrens D.L, & Williams J.T. (2019). Visualizing endogenous opioid receptors in living neurons using ligand-directed chemistry. eLife, 8, e49319.

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Planar Lipid Bilayer Electrophysiology of Opioid Peptides

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For planar lipid bilayer electrophysiology measurements, a constant voltage of −70 mV was applied over a single nanopore, and a buffer solution containing 1M KCl buffered to pH 3.8 using 50mM Citric acid titrated with bis-tris-propane was used. [Met5]-Enkephalin and [d-Ala2][d-Leu5]-Enkephalin (Sigma Aldrich) were added in 10μM concentration. Ionic currents were recorded using an Axopatch 200B amplifier coupled with a Digidata 1440a or Digidata 1550B A/D converter (Molecular Devices). All data was recorded using Clampex 10 (Molecular Devices) with a sampling frequency of 50 kHz and an analog Bessel filter of 10 kHz. Measurement procedures were first described in (Lucas et al., 2021 (link)).

de Lannoy C., Lucas F.L., Maglia G, & de Ridder D. (2021). In silico assessment of a novel single-molecule protein fingerprinting method employing fragmentation and nanopore detection. iScience, 24(10), 103202.

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