• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave the fluorogenic peptide substrate 5-FAM/QXL? 520. Peti-Peterdi, J.     et al. (2009) Physiology     24:88. The specific activity is >20 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Leu22-Arg402, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Leu22

• Predicted Molecular Mass

  43 kDa

• SDS-PAGE

  45-55 kDa, reducing conditions

Assay Procedure

Materials

• Activation Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)

• Assay Buffer: 50 mM NaH₂PO₄, 1 M NaCl, pH 7.5

• Recombinant Mouse Renin (rmRenin) (Catalog # 4277-AS)

• Trypsin (Sigma, Catalog # T-1426)

• AEBSF (Catalog # ), 100 mM stock in DMSO

• Substrate: “Rat Renin FRET” 5-FAM/QXL?520 (AnaSpec, Catalog # 62334), 100 μM stock in DMSO

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Activate rmRenin at 0.1 mg/mL with Trypsin at 2 μg/mL.

1. Dilute rmRenin to 0.2 mg/mL in Activation Buffer.

2. Dilute Trypsin to 4 μg/mL in Activation Buffer.

3. Mix equal volumes of the diluted rmRenin and Trypsin.

4. Incubate at 37 °C for 1 hour.

2. Stop Trypsin activity with 1 mM AEBSF.

1. Dilute AEBSF to 2 mM in Assay Buffer.

2. Add a volume of 2 mM AEBSF equal to the reaction volume for 1 mM AEBSF and 50 μg/mL rmRenin.

3. Incubate at room temperature for 30 minutes.

3. Dilute Substrate to 4 μM in Assay Buffer.

4. Dilute rmRenin to 2 ng/μL in Assay Buffer.

5. Load into a black well plate 50 μL of 2 ng/μL rmRenin (100 ng/well) and start the reaction by adding 50 μL of 4 μM Substrate.

6. Read at excitation and emission wavelengths of 490 nm and 520 nm (top read), respectively, in kinetic mode for 5 minutes.

7. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard calibration standard 5-FAM (AnaSpec, Catalog # 60584).

• rmRenin: 0.1 μg

• Substrate: 2 μM

Background: Renin

Renin is a aspartyl protease that plays a crucial role in the regulation of blood pressure and salt balance through the renin-angiotensin system (1). It cleaves angiotensinogen to generate angiotensin I, which can be further converted by angiotensin converting enzyme (ACE) to angiotensin II. Angiotensin II is the active molecule of the renin-angiotensin system that acts by binding to angiotensin receptors type 1 and 2 (AT-1 and AT-2), and has direct pathophysiological effects on the heart and peripheral vasculature (2). After secretion, inactive prorenin can be proteolytically activated by trypsin, cathepsin B, or other proteinases. Because of its unique specificity, inhibition of Renin is widely studied to provide selective therapy for hypertension, congestive heart failure and associated disorders (3).

• References:

1. Fuminaki, S. et al. 2004 in Handbook of Proteolytic Enzymes, Barrett, A. J. et al. eds:54.

2. Unger, T. et al. 2008 Medscape J. Med. 10:S4.

3. Dhanaraj, V. et al. 1992 Nature 357:466.

• Entrez Gene IDs:

  5972 (Human); 19701 (Mouse)

• Alternate Names:

  angiotensin-forming enzyme; Angiotensinogenase; EC 3.4.23; EC 3.4.23.15; FLJ10761; HNFJ2; REN; renin precursor, renal; Renin