Recombinant Mouse TRH-degrading Ectoenzyme/TRHDE Protein, CF

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

Recombinant Mouse TRH-degrading Ectoenzyme/TRHDE Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the pyroGlu-His bond in a fluorogenic peptide substrate, pyroGlu-His-Pro-7-amido-4-methylcoumarin (pEHP-AMC). The resulting HP-AMC was cleaved by Recombinant Human DPPIV/CD26 (Catalog # 9168-SE). The specific activity is >130 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse TRH-degrading Ectoenzyme/TRHDE protein
  Arg64-His1025, with an N-terminal 6-His tag
• Accession #: Q8K093
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Trhde
• 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: 112 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: 131 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, NaCl and Glycerol.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, 200 mM NaCl, pH 7.0
  • Recombinant Mouse TRH‑degrading Ectoenzyme/TRHDE (rmTRHDE) (Catalog # 2985-ZN)
  • Recombinant Human DPPIV/CD26 (rhCD26) (Catalog # 9168-SE)
  • Substrate: Pyr-His-Pro-AMC (Bachem, Catalog # I-1440), 2 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 rmTRHDE to 2 µg/mL in Assay Buffer.
  2. Dilute Substrate to 20 µM in Assay Buffer.
  3. Mix equal volumes of rmTRHDE and Substrate for final concentrations of 1 µg/mL and 10 µM respectively. Include a Substrate Blank containing Assay Buffer and Substrate.
  4. Incubate mixtures for 5 minutes at room temperature.
  5. Boil mixtures at 100 °C immediately after the incubation to stop the reaction.
  6. Spin down vials and gently vortex.
  7. Dilute rhCD26 to 1 µg/mL in Assay Buffer.
  8. In a plate load 50 µL of 1 µg/mL rhCD26 followed by adding 50 µL of the boiled samples to each well containing rhCD26.
  9. Incubate plate at room temperature for 10 minutes.
  10. Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, in endpoint mode.
  11. 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 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

  Per Well:
  • rmTRHDE: 0.05 µg
  • rhCD26: 0.05 µg
  • Substrate: 5 µM

Notes

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

Alternate Names for Recombinant Mouse TRH-degrading Ectoenzyme/TRHDE Protein, CF

• EC 3.4.19.6
• FLJ22381
• PAP-II
• PAP-IIthyroliberinase
• PGPEP2
• Pyroglutamyl-peptidase II
• Thyroliberinase
• thyrotropin-releasing hormone degrading enzyme
• thyrotropin-releasing hormone-degrading ectoenzyme
• TRHDE
• TRH-DE
• TRHdegrading Ectoenzyme
• TRH-degrading Ectoenzyme
• TRH-DEthyrotropin-releasing hormone degrading ectoenzyme
• TRH-specific aminopeptidase

Background

TRHDE, also known as pyroglutamyl peptidase II and thyroliberinase, is a metalloprotease that specifically removes pyroglutamate from thyrotropin-releasing hormone, a tripeptide of L-pyroglutamyl-L-histidyl-L-prolineamide. TRH functions as a hypothalamic hypophysiotropic neuropeptide and neurotransmitter/neuromodulator within the central nervous system (1). Inhibitors of TRHDE have potential applications as research and therapeutic agents because TRHDE inactivates TRH (2). TRHDE is a type II transmembrane protein and a soluble form is also present in the serum (1). The recombinant mouse TRHDE corresponds to the ectodomain of the enzyme. Its amino acid sequence is 97% and 95% identical to that of rat and human.

1. Schmitmeier, S. et al. (2002) Eur. J. Biochem. 269:1278.
2. Kelly, J.A. et al. (2005) Biochem. J. 389:569.

Bioinformatics

• Gene Symbol: Trhde
• Uniprot:
  • Q8K093()