Recombinant Human g-Glutamylcyclotransferase/CRF21, CF

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

Recombinant Human g-Glutamylcyclotransferase/CRF21, CF Summary

• Details of Functionality: Measured by its ability to release L-alanine from gamma -glutamyl-L-alanine, with the detection of alanine by alanine dehydrogenase. The specific activity is >40,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human gamma-Glutamylcyclotransferase/CRF21 protein
  Met1-Leu188
• Accession #: O75223
• N-terminal Sequence: Ala2
• Protein/Peptide Type: Recombinant Enzymes
• Gene: GGCT
• 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: 21 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: 20-22 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -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 Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, pH 9.0
  • Recombinant Human gamma ‑Glutamylcyclotransferase/CRF21 (rhCRF21) (Catalog # 5086-CT)
  • gamma -L-Glutamyl-L-alanine (Sigma, Catalog # 483834), 100 mM stock in deionized water
  • beta -Nicotinamide adenine dinucleotide ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in deionized water
  • L-Alanine Dehydrogenase (Sigma, Catalog # A7653)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhCRF21 to 0.05 ng/μL in Assay Buffer.
  2. Prepare a Substrate Mixture containing 10 mM gamma -L-Glutamyl-L-alanine, 2 mM beta -NAD, and 0.008 units/μL L‑Alanine Dehydrogenase in Assay Buffer.
  3. Load into a plate 50 μL of 0.05 ng/μL rhCRF21, and start the reaction by adding 50 μL of Substrate Mixture. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of Substrate Mixture.
  4. Read plate at a wavelength of 340 nm (bottom read) in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 6220 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD.
  
  • hCRF-21: 0.0025 μg
  • gamma -L-Glutamyl-L-alanine: 5 mM
  • beta -NAD: 1 mM
  • L-Alanine Dehydrogenase: 0.4 units

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

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

Alternate Names for Recombinant Human g-Glutamylcyclotransferase/CRF21, CF

• C7orf24
• CRF21
• gammaGlutamylcyclotransferase
• gamma-Glutamylcyclotransferase
• GCTG
• GGC
• GGCT
• LOC79017
• MGC3077

Background

Gamma-glutamylcyclotransferase (GGCT) is an essential enzyme in the gamma -glutamyl cycle that catalyzes the formation of 5-oxoproline from gamma-glutamyl dipeptides. The enzyme plays an important role in glutathione homeostasis. GGCT is also known as cytochrome c-releasing factor 21(CRF-21). It induces the release of cytochrome c from mitochondria, leading to apoptosis (1). It is a 188 amino acid protein that consists of six beta -strands, five alpha -helices and four short 3­­₁₀ helices (2). Up-regulation of the expression of GGCT has been found in various tumor tissues including lung, esophagus, stomach, bile duct, uterine cervix, colon and breast. (3, 4). Therefore, GGCT is a potential biomarker for numerous types of cancers.

1. Masuda Y. et al. (2006) Biochem. Biophys. Res. Commun. 346:454.
2. Oakley A.J. et al. (2008) J. Biol. Chem. 283:22031.
3. Gromov P. et al. (2010) J. Proteome Res. 9:3941.
4. Amano T. et al. (2012) J. Histochem. Cytochem. 60:76.

Bioinformatics

• Gene Symbol: GGCT
• Uniprot:
  • O75223(Human)