Recombinant Human Chymase/CMA1 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Chymase/CMA1 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, SUC-Ala-Ala-Pro-Phe-AMC. The specific activity is >80 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Chymase/CMA1 protein
  Met1-Asn247, with a C-terminal 10-His tag
• Accession #: P23946
• N-terminal Sequence: Gly20
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: CMA1
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 27 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 38 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in HEPES and NaCl.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Maturation Buffer: 50 mM MES, pH 5.5
  • Cathepsin Buffer: 50 mM MES, 50 mM NaCl, 5 mM DTT, pH 5.5
  • Assay Buffer: 20 mM Tris, 2 M KCl, 0.02% (v/v) Triton^® X-100, pH 9.0
  • Recombinant Human Chymase/CMA1 (rhChymase) (Catalog # 4099-SE)
  • Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
  • Heparin (Sigma, Catalog # H3393), 20 mg/mL stock in deionized water
  • N-Ethylmaleimide (NEM) (Sigma, Catalog # E1271), 50 mM stock in deionized water
  • Substrate: SUC-Ala-Ala-Pro-Phe-AMC (Bachem, Catalog # I-1465), 10 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 rhChymase to 20 µg/mL in Maturation Buffer.
  2. Dilute rmCathepsin C to 20 µg/mL in Cathepsin Buffer.
  3. Dilute Heparin to 1.5 mg/mL in deionized water.
  4. Combine 15 µL of 20 µg/mL rhChymase and 15 µL of 20 µg/mL rmCathepsin C.
  5. Add 1 µL of Heparin and mix well.
  6. Incubate at room temperature for 1 hour.
  7. Stop maturation by adding 1.98 µL of 50 mM stock of NEM for a final concentration of 3 mM.
  8. Dilute rhChymase to 2 µg/mL in Assay Buffer.
  9. Incubate at room temperature for 5 minutes.
  10. Dilute Substrate to 200 µM in Assay Buffer.
  11. Load 50 µL of 2 µg/mL rhChymase in a plate, and start the reaction by adding 50 µL of 200 µM Substrate. As a Substrate Blank combine 50 μL of Substrate with 50 μL of Assay Buffer.
  12. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  13. 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 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

  Per Well:
  • rhChymase: 0.100 µg
  • Substrate: 100 µM

Notes

Triton is a registered trademark of Union Carbide Corp.

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Chymase/CMA1 Protein, CF

• Alpha-chymase
• chymase 1 preproprotein transcript E
• chymase 1 preproprotein transcript I
• chymase 1, mast cell
• Chymase
• chymase, heart
• chymase, mast cell
• CMA1
• CYH
• CYM
• EC 3.4.21
• EC 3.4.21.39
• Mast cell protease I
• MCP3P
• Mcpt5
• MCT1
• MGC119890
• MGC119891

Background

Chymases are a group of chymotrypsin-like serine proteases secreted by mast cells (1). They are synthesized as inactive precursors containing a 2-residue propeptide, which needs to be removed by dipeptidyl peptidase I/cathepsin C for the enzymatic activity (2). Human Chymase encoded by the CMA1 gene is known to be involved in hypertention and heart failure through its ability to convert angiotensin I (Ang I) to angiotensin II (Ang II), which plays a key role in the regulation of arterial pressure (3). In addition, it is also important in physiological and pathological conditions including inflammation, fibrosis and processing of cytokines (4). Therefore, designing a specific inhibitor for Chymase activity has been a pharmacologic strategy to develop therapeutic agents.

1. Caughey, G.H. (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. ed. p. 1531, Academic Press, San Diego.
2. Murakami, M. et al. (1995) J. Biol. Chem. 270:2218.
3. Miyazaki, M. and S. Takai (2006) J. Pharmacol. Sci. 100:391.
4. Nakajima, M. and N. Naya (2002) Jpn. J. Pharmacol. 90:206.

Bioinformatics

• Gene Symbol: CMA1
• Uniprot:
  • P23946()