Recombinant Human Carboxypeptidase M Protein, CF

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

Recombinant Human Carboxypeptidase M Protein, CF Summary

• Details of Functionality: Measured by its ability to release L-arginine from Benzoyl-Ala-Arg, with detection of the arginine amino group by o-phthaldialdehyde. The specific activity is >40,000 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Carboxypeptidase M protein
  Leu18-Ser423, with a C-terminal 6-His tag
• Accession #: P14384
• N-terminal Sequence: Leu18
• Protein/Peptide Type: Recombinant Enzymes
• Gene: CPM
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Theoretical MW: 47 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: 50-56 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 MES and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Assay Procedure:
  • Assay Buffer: 50 mM MES, 0.2% Triton® X-100, 5 mM CaCl₂, pH 6.0
  • Recombinant Human Carboxypeptidase M (rhCPM) (Catalog # 7457-ZN)
  • Substrate: Bz-Ala-Arg-OH (Bachem, Catalog # G-4145), 50 mM stock in DMSO
  • 2-Mercaptoethanol (Sigma, Catalog # M7154)
  • NaOH, 2 M stock in deionized water
  • o-Phthaldialdehyde (OPA) (Sigma, Catalog # P0657), 0.373 M in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhCPM to 0.04 µg/mL in Assay Buffer.
  2. Dilute Substrate to 1 mM in Assay Buffer.
  3. Mix (in duplicate) 150 μL of 0.04 µg/mL rhCPM and 150 μL 1 mM Substrate for a final concentration of 0.02 µg/mL and 500 µM respectively. Include controls containing 150 μL of 1 mM Substrate only.
     Incubate for 10 minutes at room temperature.
  4. Stop reaction by adding 300 μL of a solution containing 15 mM o-PA in 0.2 M NaOH containing 0.1% (v/v) 2‑Mercaptoethanol and mix well.
  5. Add 150 μL of 0.04 µg/mL rhCPM to controls after stopping the reaction.
  6. Incubate all for 10 minutes at room temperature.
  7. Load 200 µL of the incubated samples in triplicate into the plate.
  8. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
  9. Calculate Specific Activity:

  Specific Activity (pmol/min/µg) = (Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)) / (Incubation time (min) x amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Derived using calibration standard L-Arginine (Sigma, Catalog # A5006).

  • rhCPM: 0.002 µg
  • Substrate: 0.25 mM
  • o-PA: 7.5 mM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd. 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 Carboxypeptidase M Protein, CF

• Carboxypeptidase M
• CPM
• EC 3.4.17
• EC 3.4.17.12

Background

Carboxypeptidase M (CPM) is a zinc metallopeptidase specific for the removal of arginine and lysine residues from peptides. CPM is bound to the plasma membrane through a glycosylphosphatidylinositol anchor (1). The enzyme is thought to regulate the actions of some peptide hormones at the cell surface through their degradation (2). CPM is a biomarker for well-differentiated liposarcomas (3) and is also a marker for the maturation of monocytes to macrophages (4). CPM binds to the kinin B1 receptor on the cell surface, forming a complex that potentiates the signaling ability of the kinin B1 receptor (5).  Recombinant human CPM was expressed as a C-terminally truncated protein to prevent the formation of the glycosylphosphatidylinositol anchor, resulting in the secretion of the protein.

1. Tan F. et al. (2003) Biochem. J. 370:567.
2. Reverter D. et al. (2004) J. Mol. Biol. 338:257.
3. Erickson-Johnson M.R. et al. (2009) Mod. Pathol. 22:1541.
4. Rehli M. et al. (2000) Adv. Exp. Med. Biol. 477:205.
5. Zhang X. et al. (2011) J. Biol. Chem. 286:18547.

Bioinformatics

• Gene Symbol: CPM
• Uniprot:
  • P14384(Human)