Recombinant Human ECE-2 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human ECE-2 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPPGFSAFK(Dnp)-OH (Catalog # ES005). The specific activity is > 250 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human ECE-2 protein
  Gly104-Trp787, with an N-terminal 10-His tag and Val
• Accession #: EAW78275
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: ECE2
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Theoretical MW: 79 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 113 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and ZnCl₂.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 0.1 M MES, 0.25 M NaCl, pH 5.75
  • Recombinant Human ECE-2 (rhECE-2) (Catalog # 1645-ZN)
  • Substrate: MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH (Catalog # ES005)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhECE-2 to 100 µg/mL in Assay Buffer.
  2. Incubate at room temperature for 30 minutes.
  3. Dilute the incubated rhECE-2 to 0.8 ng/µL.
  4. Dilute Substrate to 20 µM in Assay Buffer.
  5. Load 50 µL of 0.8 ng/µL rhECE-2 into a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing Assay Buffer and 20 µM Substrate.
  6. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  7. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank

  **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

  Per Well:
  • rhECE-2: 0.040 µg
  • Substrate: 10 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human ECE-2 Protein, CF

• EC 3.4.24
• EC 3.4.24.71
• ECE2
• ECE-2
• endothelin converting enzyme 2
• endothelin-converting enzyme 2
• KIAA0604MGC2408
• MGC17664
• MGC78487

Background

ECE-2 is a zinc protease of the neprilysin (NEP) family, which also includes ECE-1, PEX, XCE, DINE, Kell and several NEP-like proteins (1). ECE-2 is a type II transmembrane protein with a short cytoplasmic tail and a large ectodomain. Three alternatively spliced forms differ in their cytoplasmic tail (2). In addition to big endothelin-1, ECE-2 cleaves a variety of bioactive peptides such as bradykinin, neurotensin, angiotensin I, substance P, dynorphin B, proenkephalin-derived peptides such as peptide E, BAM 22 and BAM18, and PEN-LEN, an endogenous inhibitor of prohormone convertase 1 (3). Together with ECE-1, it is also involved in degradation of beta -amyloid peptide (4). The ectodomain of human ECE-2 was expressed with an N-terminal His tag and purified.

1. Turner, A.J. et al. (2001) BioEssays 23:261.
2. Lorenzo, M.N. et al. (2001) Biochim. Biophys. Acta 1522:46.
3. Mzhavia, N. et al. (2003) J. Biol. Chem. 278:14704.
4. Eckman, E.A. et al. (2003) J. Biol. Chem. 278:2081.

Publications for ECE-2 (1645-ZN)(3)

Publications using 1645-ZN

• JS Miners, S Love Endothelin-converting enzymes degrade ?-synuclein and are reduced in dementia with Lewy bodies J. Neurochem, 2017;0(0):. 2017 [PMID: 28171705] (Bioassay)
• Palmer JC, Baig S, Kehoe PG, Love S Endothelin-converting enzyme-2 is increased in Alzheimer's disease and up-regulated by Abeta. Am. J. Pathol., 2009;175(1):262-70. 2009 [PMID: 19541930] (ELISA (Standard), N/A)
• Roosterman D, Kempkes C, Cottrell GS, Padilla BE, Bunnett NW, Turck CW, Steinhoff M Endothelin-converting enzyme-1 degrades internalized somatostatin-14. Endocrinology, 2008;149(5):2200-7. 2008 [PMID: 18276747] (Enzyme Assay, Human)

Bioinformatics

• Gene Symbol: ECE2
• Uniprot:
  • EAW78275()