Recombinant Cynomolgus/Rhesus Neprilysin/CD10 His Protein CF

• Assay Buffer: 50 mM Tris, 0.5 M NaCl, pH 9.0
  
• Recombinant Cynomolgus/Rhesus Neprilysin (rcyno/rhesusNeprilysin) (Catalog # 10834-ZN)
• Substrate:  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)
• Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rcyno/rhesusNeprilysin to 0.2 µg/mL in Assay Buffer.
2. Dilute Substrate to 60 µM in Assay Buffer.
3. Load into a plate 50 µL of 0.2 µg/mL rcyno/rhesusNeprilysin and start the reaction by adding 50 µL of 60 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 60 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:

• rcyno/rhesusNeprilysin: 0.01 µg
• Substrate: 30 µM
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  

Scientific Data

Neprilysin/CD10, also known as NEP, neutral endopeptidase, enkephalinase, and common acute lymphocytic leukemia antigen (CALLA), is a zinc-dependent metallopeptidase type II integral membrane protein of the M13 subfamily of neutral endopeptidases. The enzyme functions both as an endopeptidase with a thermolysin-like specificity and as a dipeptidylcarboxypeptidase. It cleaves and degrades a variety of bioactive peptides including natriuretic peptides, enkephalins, and tachykinins at the amino side of hydrophobic residues. Neprilysin is widely distributed in tissues and highly conserved among mammals (1). Neprilysin is a noncovalently associated homodimer where the monomer is composed of a short intracellular domain, transmembrane helix, and large C-terminal extracellular domain including two domains that form a central cavity containing the active site (2). Although a membrane protein, an alternatively processed soluble form has been documented in circulation and retains similar enzymatic activity to the membrane bound Neprilysin (3). The soluble form is of interest as a prognostic biomarker for heart failure (4,5) and inhibition of Neprilysin is a therapeutic strategy for heart failure (6). Neprilysin was found to be a major degrading enzyme of amyloid beta peptide (A beta) in the brain, making it an important target in Alzheimer's disease studies (7). It metabolizes sensory and inflammatory neuropeptides making it a target for pain perception (8) and is an important marker for some types of lymphomas (9,10). There is also interest in Neprilysin as a biomarker (11) and as a therapeutic inhibition target for treatment of type 2 diabetes (12).