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
>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
Dilutions
Enzyme Activity
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
Publications
Read Publications using 1645-ZN in the following applications:
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rhECE-2 to 100 µg/mL in Assay Buffer.
Incubate at room temperature for 30 minutes.
Dilute the incubated rhECE-2 to 0.8 ng/µL.
Dilute Substrate to 20 µM in Assay Buffer.
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.
Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
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.
Turner, A.J. et al. (2001) BioEssays 23:261.
Lorenzo, M.N. et al. (2001) Biochim. Biophys. Acta 1522:46.
Mzhavia, N. et al. (2003) J. Biol. Chem. 278:14704.
Eckman, E.A. et al. (2003) J. Biol. Chem. 278:2081.
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