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.
Recombinant Mouse TRH-degrading Ectoenzyme/TRHDE Protein, CF
R&D Systems | Catalog # 2985-ZN
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Key Product Details
- R&D Systems NS0-derived Recombinant Mouse TRH-degrading Ectoenzyme/TRHDE Protein (2985-ZN)
- Quality control testing to verify active proteins with lot specific assays by in-house scientists
- All R&D Systems proteins are covered with a 100% guarantee
Source
NS0
Accession Number
Applications
Enzyme Activity
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Product Specifications
Source
Mouse myeloma cell line, NS0-derived mouse TRH-degrading Ectoenzyme/TRHDE protein
Arg64-His1025, with an N-terminal 6-His tag
Arg64-His1025, with an N-terminal 6-His tag
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
N-terminal Sequence Analysis
His
Predicted Molecular Mass
112 kDa
SDS-PAGE
131 kDa, reducing conditions
Activity
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.
The specific activity is >130 pmol/min/µg, as measured under the described conditions.
Formulation, Preparation, and Storage
2985-ZN
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Background: TRH-degrading Ectoenzyme/TRHDE
References
- Schmitmeier, S. et al. (2002) Eur. J. Biochem. 269:1278.
- Kelly, J.A. et al. (2005) Biochem. J. 389:569.
Long Name
Thyrotropin Releasing Hormone-degrading Ectoenzyme
Alternate Names
PAP-II, PGPEP2, Thyroliberinase, TRH-DE, TRHDE, TRHdegrading Ectoenzyme
Gene Symbol
TRHDE
UniProt
Additional TRH-degrading Ectoenzyme/TRHDE Products
Product Documents for Recombinant Mouse TRH-degrading Ectoenzyme/TRHDE Protein, CF
Certificate of Analysis
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Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Mouse TRH-degrading Ectoenzyme/TRHDE Protein, CF
For research use only
Related Research Areas
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Protocols
View specific protocols for Recombinant Mouse TRH-degrading Ectoenzyme/TRHDE Protein, CF (2985-ZN):
Materials
- 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
- Dilute rmTRHDE to 2 µg/mL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- 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.
- Incubate mixtures for 5 minutes at room temperature.
- Boil mixtures at 100 °C immediately after the incubation to stop the reaction.
- Spin down vials and gently vortex.
- Dilute rhCD26 to 1 µg/mL in Assay Buffer.
- 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.
- Incubate plate at room temperature for 10 minutes.
- Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, in endpoint mode.
- 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
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