Recombinant Mouse ECE-1 Protein, CF Summary
Product Specifications
Gln89-Trp769, with and N-terminal 6-His tag, Gln89-Trp769
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
5796-ZN
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and ZnCl2. |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
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
- Dilute rmECE-1 to 0.1 µg/mL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- 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.
- 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:- rmECE-1: 5 ng
- Substrate: 10 µM
Reconstitution Calculator
Background: ECE-1
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.
- Yanagisawa, M. et al. (1998) Nature 332:411.
- Turner, A.J. et al. (2001) BioEssays 23:261.
- Johnson, G.D. et al. (1999) J. Biol. Chem. 274:4053.
- Cottrell, G.S. et al. (2009) J. Biol. Chem. 284:22411.
- Lindenau, S. et al. (2006) Gene 373:109.
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