Recombinant Mouse ECE-1 Protein, CF

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Product Details

Summary
Reactivity MuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

Order Details

Recombinant Mouse ECE-1 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 >2,250 pmol/min/μg, as measured under the described conditions. 
Source
Chinese Hamster Ovary cell line, CHO-derived mouse ECE-1 protein
Gln89-Trp769, with and N-terminal 6-His tag, Gln89-Trp769
Accession #
N-terminal Sequence
Gln89
Structure / Form
Disulfide-linked dimer
Protein/Peptide Type
Recombinant Enzymes
Purity
>90%, 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
78 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
90-130 kDa, reducing conditions

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in Tris, NaCl and ZnCl2.
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
Assay Procedure
  • Assay Buffer: 50 mM MES, 0.1 M NaCl, pH 6.0
  • Recombinant Human ECE-1 (rhECE-1) (Catalog # 5796-ZN)
  • Fluorogenic Peptide Substrate V: MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH (Catalog # ES005), 2 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmECE-1 to 0.1 µg/mL in Assay Buffer.
  2. Dilute Substrate to 20 µM in Assay Buffer.
  3. Load into a black well plate 50 µL of 0.1 µg/mL of rmECE-1, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
  4. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  5. 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:
  • rmECE-1: 5 ng
  • Substrate: 10 µM

Notes

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

Alternate Names for Recombinant Mouse ECE-1 Protein, CF

  • EC 3.4.24
  • EC 3.4.24.71
  • ECE
  • ECE1
  • ECE-1
  • endothelin converting enzyme 1
  • endothelin-converting enzyme 1

Background

Endothelin-converting enzymes (ECEs) hydrolyze a specific peptide bond of big endothelins to produce active endothelins, some of the most potent vasoconstrictors known (1). ECE-1 is a member of the M13 zinc metallopeptidase family. Other members of the M13 family include thermolysin, neprilysin, Kell, and ECE-2 (2). M13 metallopeptidases can be distinguished from other metallopeptidases by their sensitivity to inhibition by phosphoramidon. ECE-1 is most highly expressed in the cardiovascular endothelium, but is also expressed in some endocrine tissues (3). ECE-1 is known to hydrolyze a variety of bioactive peptides, including bradykinin, neurotensin, angiotensins, and Substance P, with a substrate specificity similar to that of neprilysin (4). ECE-1 displays pronounced pH dependence in its substrate specificity (5). The degradation of Substance P by ECE-1 in endosomes regulates beta -arrestin-dependent ERK-2 signaling to prevent cell death in some neuronal cells (6). Four isoforms of ECE-1 are present in humans and mice, all of which encode a Type II integral membrane protein (7). The four isoforms share a common extracellular catalytic domain, differing in their N-terminal cytoplasmic tail regions. The recombinant mouse ECE-1 transmembrane and cytoplasmic tail domains were replaced with a signal sequence, resulting in the secretion of the soluble catalytic ectodomain.

  1. Yanagisawa, M. et al. (1998) Nature 332:411.
  2. Turner, A.J. et al. (2001) BioEssays 23:261.
  3. Davenport, A.P. et al. (1998) Histochem. J. 30:359.
  4. Johnson, G.D. et al. (1999) J. Biol. Chem. 274:4053.
  5. Fahnoe, D.C. et al. (2000) J. Cardiovasc. Pharmacol. 36:S22.
  6. Cottrell, G.S. et al. (2009) J. Biol. Chem. 284:22411.
  7. Lindenau, S. et al. (2006) Gene 373:109.  

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