Recombinant Human PRCP Protein, CF

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

Recombinant Human PRCP Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the synthetic peptide substrate N-benzyloxycarbonyl-Pro-Ala (Z-Pro-Ala). The specific activity is >2,500 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Lysosomal Pro-X Carboxypeptidase/PRCP protein
  Met1-His496, with a C-terminal 6-His tag
• Accession #: P42785
• N-terminal Sequence: Leu22
• Protein/Peptide Type: Recombinant Enzymes
• Gene: PRCP
• 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

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 54 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: 60-70 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 Sodium Acetate, 0.1 M NaCl, pH 4.0
  • Recombinant Human Lysosomal Pro‑X Carboxypeptidase/PRCP (rhPRCP) (Catalog # 7164-SE)
  • Substrate: Z-Pro-Ala (Bachem, Catalog # C-2485), 100 mM stock in deionized water
  • 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 rhPRCP to 0.2 µg/mL in Assay Buffer.
  2. Dilute Substrate to 400 µM in Assay Buffer.
  3. Mix 125 μL of 0.2 µg/mL rhPRCP and 125 μL 400 µM Substrate for a final concentration of 0.1 µg/mL and 200 µM respectively. Include a control containing 125 μL of 0.2 µg/mL rhPRCP that has been heat treated for 5 minutes at 100 °C and 125 μL 400 µM Substrate.
  4. Incubate for 30 minutes at 37 °C.
  5. Stop reaction by adding 250 μL of a solution containing 15 mM o-PA in 0.2 M NaOH containing 0.1% (v/v) 2-Mercaptoethanol.
  6. Incubate for 10 minutes at room temperature.
  7. Load 200 µL of the incubated samples in duplicate 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 heat treated control
  **Derived using calibration standard L-Alanine (Sigma, Catalog # A7469).

  Per Well:
  • rhPRCP: 0.01 µg
  • Substrate: 0.1 mM

Notes

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

Alternate Names for Recombinant Human PRCP Protein, CF

• Angiotensinase C
• EC 3.4.16.2
• HUMPCP
• Lysosomal carboxypeptidase C
• lysosomal Pro-X carboxypeptidase
• PCPMGC2202
• PRCP
• Proline carboxypeptidase
• prolylcarboxypeptidase (angiotensinase C)
• prolylcarboxypeptidase isoform 1 preproprotein
• Prolylcarboxypeptidase

Background

Lysosomal Pro-X Carboxypeptidase (PRCP) is a serine carboxypeptidase found principally in lysosomes, but can also be secreted or associated with the plasma membrane. The enzyme is specific for peptide substrates with a penultimate proline residue. PRCP is also known as Angiotensinase C because of its activity against angiotensin II and angiotensin III (1). PRCP is also known to be involved in the activation of prekallikrein on the surface of endothelial cells (2). Because of these activities, PRCP is considered to be a cardioprotective enzyme (3). PRCP has been shown to degrade the bioactive peptide a-melonocyte stimulating hormone, thereby destroying its activity (4). Therefore PRCP may play a role in the melanocortin signaling pathway and the regulation of energy metabolism (5).

1. Yang, H.Y.T. et al. (1968) Nature 218:1224.
2. Shariat-Madar, Z. et al. (2002) J. Biol. Chem. 277:17962.
3. Mallela, J. et al. (2009) Int. J. Biochem. Cell Biol. 41:477.
4. Wallingford, N. et al. (2009) J. Clin. Invest. 119:2291.
5. Diano, S. et al. (2011) Front. Neuroendocrinol. 32:70.

Publications for Lysosomal Pro-X Carboxypeptidase/PRCP (7164-SE)(1)

Publication using 7164-SE

• DR Dunshee, TW Bainbridge, NM Kljavin, J Zavala-Sol, AC Schroeder, R Chan, R Corpuz, M Wong, W Zhou, G Deshmukh, J Ly, DP Sutherlin, JA Ernst, J Sonoda Fibroblast Activation Protein Cleaves and Inactivates Fibroblast Growth Factor 21 J. Biol. Chem, 2016-01-21;291(11):5986-96. 2016-01-21 [PMID: 26797127]

Bioinformatics

• Gene Symbol: PRCP
• Uniprot:
  • P42785()