Recombinant Mouse Mast Cell Protease-1/Mcpt1 Protein, CF

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

Recombinant Mouse Mast Cell Protease-1/Mcpt1 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave Suc-Ala-Ala-Pro-Phe-AMC (Suc-AAPF-AMC, Bachem, Catalog # I-1465). The specific activity is >35 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Mast Cell Protease-1/Mcpt1 protein
  Glu19-Lys246, with a C-terminal 10-His tag
• Accession #: P11034
• N-terminal Sequence: Glu19
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Mcpt1
• Purity: >95%, 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: 33 kDa, 35 kDa and 36 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 Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Activation Buffer: 50 mM MES, 50 mM NaCl, 5 mM DTT, pH 5.5
  • Assay Buffer: 50 mM Tris, 2 M NaCl, 0.025% (w/v) Brij-35, pH 8.0
  • Recombinant Mouse Mast Cell Protease‑1/Mcpt1 (rmMcpt1) (Catalog # 5146-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 rmCathepsin C to 120 µg/mL in Activation Buffer.
  2. Dilute rmMcpt1 to 200 µg/mL in Activation Buffer.
  3. Dilute Heparin to 160 µg/mL in Activation Buffer.
  4. Combine 15 µL of 200 µg/mL rmMcpt1, 15 µL of 120 µg/mL rmCathepsin C and 30 µL of 160 µg/mL Heparin. Mix well.
  5. Incubate reaction mixture for 2 hours at 37 °C.
  6. Dilute NEM to 10 mM in Assay Buffer.
  7. Stop activation by adding 60 µL of 10 mM NEM.
  8. Dilute activated rmMcpt1 to 8 ng/µL in Assay Buffer.
  9. Dilute Substrate to 200 µM in Assay Buffer.
  10. Load into a plate 50 µL of 8 ng/µL rmMcpt1, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 200 µM Substrate.
  11. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.

  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 (AMC) (Sigma, Catalog # A-9891).

  Per Well:
  • rmMcpt1: 0.4 µg
  • Substrate: 100 µM

Notes

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

Alternate Names for Recombinant Mouse Mast Cell Protease-1/Mcpt1 Protein, CF

• AV080368
• Mast Cell Protease1
• Mast Cell Protease-1
• Mcp1
• Mcp-1
• MMPC-1

Background

Mast cell protease-1 (Mcpt1), also known as beta ‑chymase, is a member of the Chymase family of chymotrypsin-like serine proteases (1). mMcpt1 is a mast cell protease predominantly expressed in intestinal mucosal mast cells where it promotes mucosal permeability in intestinal allergic hypersensitivity reactions (2). Its activation is completed by the removal of a two residue N‑terminal propeptide by a dipeptidyl peptidase (Cathepsin C) (3). Like human alpha ‑Chymase, Mcpt1 is capable of the conversion of angiotensin I to angiotensin II, which plays a key role in the regulation of arterial pressure (4). Studies have shown that specific chymase inhibitors are able to diminish the development of abdominal aortic aneurysm and reduce the adhesion formation after cardiac surgery in hamsters (5, 6). Therefore, the development of specific inhibitors of chymase activity has been a pharmacologic strategy to develop therapeutic agents.

1. Caughey, G.H. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.), p. 1531 Academic Press, San Diego.
2. Wastling, J.M. et al. (1998) Am. J. Pathol. 153:491.
3. Murakami, M. et al. (1995) J. Biol. Chem. 270:2218.
4. Saito, K. et al. (2003) Biochem. Biophys. Res. Commun. 302:773.
5. Tsunemi, K. et al. (2004) J. Pharmacol. Exp. Ther. 309:879.
6. Soga, Y. et al. (2004) J. Thorac. Cardiovasc. Surg. 127:72.

Publications for Mast Cell Protease-1/Mcpt1 (5146-SE)(1)

Publication using 5146-SE

• Zhu, A;Baur, C;Götz, P;Elbs, K;Lasch, M;Faro, A;Preissner, KT;Deindl, E; The Complement System Is Essential for Arteriogenesis by Enhancing Sterile Inflammation as a Relevant Step in Collateral Artery Growth Cells 2024-08-23 [PMID: 39272977] (In vivo assay, Mouse)

Bioinformatics

• Gene Symbol: Mcpt1
• Uniprot:
  • P11034()