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Recombinant Mouse Carboxypeptidase A4/CPA4 Protein, CF

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Product Details

Summary
Reactivity MuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Mouse Carboxypeptidase A4/CPA4 Protein, CF Summary

Details of Functionality
Measured by its ability to cleave the colorimetric peptide substrate Ac-Phe-Thiaphe-OH in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB). Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468. The specific activity is >400 pmol/min/µg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived mouse Carboxypeptidase A4/CPA4 protein
Gly17-Tyr420, with a C-terminal 10-His tag
Accession # Q6P8K8
Accession #
N-terminal Sequence
Gly17
Protein/Peptide Type
Recombinant Enzymes
Gene
Cpa4
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
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
48 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 Tris, 10 mM CaCl2, 150 mM NaCl , 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Assay Buffer: 50 mM Tris, pH 8.0
  • Recombinant Mouse Carboxypeptidase A4/CPA4 (rmCPA4) (Catalog # 2899-ZN)
  • Trypsin (Sigma, Catalog # T-1426)
  • AEBSF (Catalog # EI001), 100 mM in deionized water
  • Substrate: Ac-Phe-Thiaphe-OH (Peptides International, Catalog # STP-3621-PI),10 mM stock in DMSO
  • 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB), (Sigma, Catalog # D-8130), 10 mM stock in DMSO
  • 96 well clear plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rmCPA4 to 100 µg/mL with 1.0 µg/mL Trypsin in Activation Buffer.
  2. Incubate at room temperature for 1 hour.
  3. Stop Trypsin activity by adding AEBSF at a final concentration of 1 mM.
  4. Incubate at room temperature for 15 minutes.
  5. Dilute active rmCPA4 to 6 µg/mL in Assay Buffer.
  6. Dilute Substrate to 200 µM with 200 µM DTNB in Assay Buffer.
  7. Load 50 µL of the 6 µg/mL rmCPA4 into a plate, and start the reaction by adding 50 µL of Substrate/DTNB mixture. Include a Substrate Blank with 50 µL of Assay Buffer and 50 µL of Substrate/DTNB mixture.
  8. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
  9. Calculate specific activity using the following formula:

     Specific Activity (pmol/min/µg) =

 Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)

     *Adjusted for Substrate Blank 
     **Using the extinction coefficient 13260 M-1cm-1 
     ***Using the path correction 0.32 cm
     Note: the output of many spectrophotometers is in mOD. Per Well:
  • rmCPA4: 0.3 µg
  • Substrate: 100 µM
  • DTNB: 100 µM

Notes

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

Alternate Names for Recombinant Mouse Carboxypeptidase A4/CPA4 Protein, CF

  • Carboxypeptidase A3
  • Carboxypeptidase A4
  • CPA3EC 3.4.17
  • CPA4
  • EC 3.4.17.-
  • EC 3.4.17.1

Background

Carboxypeptidase A4 encoded by the CPA4 gene is a member of the metallocarobxypeptidase family that cleaves the C-terminal amide or ester bond of peptides that have a free C-terminal carboxyl group. The deduced amino acid sequence of mouse CPA4 consists of a signal peptide (residues 1 to 16), a pro region (residue 17 to 113), and a mature chain (residues 114 to 420). The purified recombinant mouse CPA4 corresponds mainly to the pro form, which can be activated and assayed under the conditions described in the Activity Assay Protocol.

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Bioinformatics

Gene Symbol Cpa4
Uniprot