详细说明
- Purity>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
- Endotoxin Level<0.01 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured by its ability to cleave the fluorogenic peptide substrate, Mca-RPPGFSAFK(Dnp)-OH (Catalog # ). The specific activity is >3,000 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com
- SourceChinese Hamster Ovary cell line, CHO-derived Tyr52-Trp750, with an N-terminal 5-His tag
- Accession #
- N-terminal Sequence
AnalysisHis
- Predicted Molecular Mass81 kDa
- SDS-PAGE97 kDa, under reducing conditions
| 1126-ZNC | | |
| Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and ZnCl 2. | ||
| Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. | ||
| Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
- Assay Buffer: 50 mM Tris, pH 9.0
- Recombinant Mouse Neprilysin (rmNeprilysin) (Catalog # 1126-ZNC)
- Substrate: MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH (Catalog # ), 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 rmNeprilysin to 0.1 µg/mL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- In a plate, load 50 µL of 0.1 µg/mL rmNeprilysin, 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).
- rmNeprilysin: 0.005 µg
- Substrate: 10 µM
Neprilysin, (NEP, neutral endopeptidase 24.11, EC 3.4.24.11), is a zinc metallopeptidase expressed at the cell surface of a variety of cells. The enzyme functions both as an endopeptidase with a thermolysin-like specificity and as a dipeptidylcarboxypeptidase. NEP has been shown to be involved in the degradation of enkephalins in the mammalian brain and the inactivation of circulating atrial natriuretic peptide (1, 2). NEP has also been identified as the common acute lymphocytic leukemia antigen (CALLA), and is expressed on the surface of lymphocytes in some disease states (3, 4). These and other observations have resulted in considerable interest in NEP as a target for analgesics and antihypertensive drugs. NEP is also a major degrading enzyme of amyloid beta peptide (A beta ) in the brain, indicating that down-regulation of NEP activity, which could be caused by aging, can contribute to the development of Alzheimer's disease by promoting A beta accumulation (5).
- References:
- Malfroy, B. et al. (1978) Nature 276:523.
- Kenny, A.J. and Stephenson, S.L. (1988) FEBS Lett. 232:1.
- LeTarte, M. et al. (1988) J. Exp. Med. 168:1247.
- Shipp, M.A. et al. (1988) Proc. Natl. Acad. Sci. USA 85:4819.
- Itwata, N. et al. (2001) Science 292:1550.
-
- Entrez Gene IDs:4311 (Human); 17380 (Mouse); 24590 (Rat)
- Alternate Names:Atriopeptidase; CALLA; CALLAmembrane metallo-endopeptidase (neutral endopeptidase, enkephalinase); CD10 antigen; CD10; CD10); CD10membrane metallo-endopeptidase variant 1; Common acute lymphocytic leukemia antigen; DKFZp686O16152; EC 3.4.24; EC 3.4.24.11; Enkephalinase; EPN; Leu-19; membrane metallo-endopeptidase variant 2; membrane metallo-endopeptidase; MGC126681; MGC126707; MME; NEPmembrane metallo-endopeptidase (neutral endopeptidase, enkephalinase, CALLA; Neprilysin; Neutral Endopeptidase 24.11; Neutral endopeptidase; NKH1; SFE; Skin fibroblast elastase
400-6226-992