Recombinant Human GGT5 Protein, CF

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

Recombinant Human GGT5 Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze glutathione to Glu and Cys-Gly. The specific activity is >265 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human GGT5 protein
  Ser30-Tyr586, with an N-terminal 6-His tag.
• Accession #: P36269
• N-terminal Sequence: His and Thr388
• Protein/Peptide Type: Recombinant Enzymes
• Gene: GGT5
• 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: 60 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: 24-27(mature), 41-46 (mature) & 64-66 (pro) 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 Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 0.05% Tween® 20, pH 8.0
  • Recombinant Human GGT5 (rhGGT5) (Catalog # 7209-GT)
  • Recombinant Human NQO-1 (rhNQO-1) (Catalog # 7567-DH)
  • Glutamate dehydrogenase (GIDH) (Sigma, Catalog # G7882), 200 U/mL stock in 50 mM Tris, 0.05% Tween 20, pH 8.0
  • Nicotinamide adenine dinucleotide ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in deionized water
  • Resazurin (Catalog # AR002)
  • Glutathione, reduced (GSH) (Amresco, Catalog # 399), 250 mM stock in deionized water
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhGGT5 to 1 µg/mL in Assay Buffer.
  2. Prepare substrate mixture containing 2 mM GSH, 100 U/mL GIDH, 2 mM beta -NAD, 40 µM Resazurin, and 4 µg/mL rhNQO-1 in Assay Buffer.
  3. Load 50 µL rhGGT5 into wells of plate. Include 50 µL Assay Buffer for a Substrate Blank.
  4. Start reaction by adding 50 µL substrate mixture to all wells.
  5. Read the plate at excitation and emission wavelengths of 540 and 585 nm, respectively, for five minutes in kinetic mode.
  6. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Derived using calibration standard Resorufin (Sigma, Catalog # R3257)

  Per Well:
  • rhGGT5: 0.050 µg
  • GSH: 1 mM
  • GIDH: 5 U
  • beta -NAD: 1 mM
  • rhNQO-1: 0.2 μg
  • Resazurin: 0.02 mM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd. Tween is a registered trademark of ICI Americas.

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

Alternate Names for Recombinant Human GGT5 Protein, CF

• DKFZp566O011
• EC 2.3.2.2
• gamma-glutamyl cleaving enzyme
• Gamma-glutamyl transpeptidase-related enzyme
• gamma-glutamyl transpeptidase-related protein
• gamma-glutamyltransferase 5
• Gamma-glutamyltransferase-like activity 1FLJ92733
• Gamma-glutamyltranspeptidase 5
• GGT 5
• GGT5
• GGTLA1
• GGT-rel

Background

Gamma-glutamyl transpeptidase-5 (GGT5) is expressed as a pro form, which undergoes autocatalytic cleavage in the endoplasmic reticulum to become the two chain mature form (1). The mature form of GGT is a membrane protein embedded into the outer surface of the plasma membrane through its N-terminal transmembrane domain. It cleaves the gamma-glutamyl peptide bond of glutathione (GSH) and glutathione S-conjugates and transfers the g-glutamyl moiety to acceptors (2). By breaking down extracellular GSH into its constitutive amino acids, its activity is critical for maintaining GSH and cysteine homeostasis. GGT1 and GGT5 are the most fully characterized members of the GGT family (2). Elevated activity of GGT1 in blood has been used to measure liver damage such as that found in hepatocellular carcinoma or alcoholic liver (3). GGT5, also known as g-glutamyl leukotrienase (GGL), may play an important role in leukotriene D4 synthesis and in inflammatory processes.

1. Kinlough C.L. et al. (2005) Methods Enzymol. 401:426.
2. Heisterkamp N. et al. (2008) Hum. Genet. 123:321.
3. Whitfield J.B. (2001) Crit. Rev. Clin. Lab. Sci. 38:263.
4. Han B. et al. (2002) Am. J. Path. 161:481.

Bioinformatics

• Gene Symbol: GGT5
• Uniprot:
  • P36269(Human)