• 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.  

• Activity

  Measured 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

• Source

  Chinese Hamster Ovary cell line, CHO-derived Tyr52-Trp750, with an N-terminal 5-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  His    
   

• Predicted Molecular Mass

  81 kDa

• SDS-PAGE

  97 kDa, under reducing conditions

Assay Procedure

Materials

• 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

1. Dilute rmNeprilysin to 0.1 μg/mL in Assay Buffer.

2. Dilute Substrate to 20 μM in Assay Buffer.

3. 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.

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:

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

Per Well:

• rmNeprilysin: 0.005 μg

• Substrate: 10 μM

Background: Neprilysin/CD10

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:

1. Malfroy, B.         et al. (1978) Nature         276:523.

2. Kenny, A.J. and Stephenson, S.L. (1988) FEBS Lett. 232:1.

3. LeTarte, M. et al. (1988) J. Exp. Med. 168:1247.

4. Shipp, M.A. et al. (1988) Proc. Natl. Acad. Sci. USA 85:4819.

5. 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