Recombinant Human GGT1 His-tag Protein, CF

Recombinant Human GGT1 His-tag (Catalog # 10977-GT) is measured by its ability to hydrolyze glutathione to Glu and Cys-Gly.

2 μg/lane of Recombinant Human GGT1 His-tag Protein (Catalog # 10977-GT) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 56-69 kDa and 22-26 kDa, under reducing conditions.

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

Recombinant Human GGT1 His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze glutathione to Glu and Cys-Gly. The specific activity is >1300 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human gamma-Glutamyltransferase 1/GGT1 protein
  Pro27-Tyr569 with a C-terminal 6-His
• Accession #: P19440.2
• N-terminal Sequence: Pro27 & Thr381
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 39 kDa (heavy chain) & 21 kDa (light chain).
  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: 56-69 kDa & 22-26 kDa, under 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 visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 0.05% Tween® 20, pH 8.0
  • Recombinant Human GGT-1 (rhGGT-1) (Catalog # 10977-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 M5 by Molecular Devices) or equivalent
  1. Dilute rhGGT-1 to 0.4 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 in a plate 50 µL of 0.4 µg/mL rhGGT-1, and start the reaction by adding 50 µL of substrate mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of substrate mixture.
  4. Read at excitation and emission wavelengths of 540 and 585 nm, respectively, for five minutes in kinetic mode.
  5. 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:
  • rhGGT-1: 0.020 µg
  • GSH: 1 mM
  • GIDH: 5 U
  • beta -NAD: 1 mM
  • rhNQO-1: 0.2 µg
  • Resazurin: 0.02 mM

Notes

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

Alternate Names for Recombinant Human GGT1 His-tag Protein, CF

• CD224 antigen
• CD224
• D22S672
• D22S73
• D22S732
• EC 2.3.2.2
• gamma-glutamyl transpeptidase
• gamma-Glutamyltransferase 1
• gamma-glutamyltransferase 1MGC96904
• gamma-glutamyltranspeptidase 1
• GGT 1
• GGT
• GGT-1
• GGTD
• GGTMGC96963
• glutamyl transpeptidase
• GTG
• MGC96892

Background

Gamma-glutamyl transferase 1 (GGT1), also known as Gamma-glutamyl transpeptidase 1, is a ubiquitous enzyme that cleaves the gamma-glutamyl bond substrates where the glutamate moiety is available. This makes GGT1 crucial for metabolism of glutathione, the most important cellular antioxidant in humans and for its role in leukotriene biosynthesis, required for lipid metabolism. Although GGT1 comes from a 7 member multigene family, only GGT1 and GGT5 have enzymatic activity (1, 2). GGT5 has 40% amino acid homology with GGT1 and only 4% of the activity (2, 3) making GGT1 the primary contributor to enzymatic activity in the cells. GGT1 is synthesized as a single 569 amino acid polypeptide that is autocleaved by threonine 381 into a mature, activated, heterodimeric glycoprotein composed of a large subunit with an N-terminal hydrophobic transmembrane domain that anchors the enzyme to the membrane and a small catalytic subunit containing the catalytic nucleophile threonine 381 (4, 5). The human GGT1 active site is located in a deep channel but remains in an open configuration (4). It has a unique buried chloride ion that is integral to the protein structure (4). GGT1 contributes to cysteine homeostasis, intracellular redox, and inflammation (2, 6-8) and has been implicated to play a role in Parkinson and cardiovascular disease (5, 9), is a diagnostic marker of several diseases including liver and pancreatic cancer and is induced in several cancers to contribute to resistance to chemotherapeutic agents (7, 10-14). 

1. Pawlak, A. et al. (1988) J. Biol. Chem. 263:9913.
2. Wickham, S. et al. (2011) Anal. Biochem. 414:208.
3. Heisterkamp, N. et al. (1991) Proc. Natl. Acad. Sci. USA 88:6303.
4. West, M.B. et al. (2013) J Biol. Chem. 288:31902.
5. Ndrepepa, G. et al. (2018) Clin. Chim. Acta. 476:130.
6. Lieberman, M.W. et al. (1996) Proc. Natl. Acad. Sci. 93: 7923.
7. Hanigan, M.H. (2014) Adv. Cancer Res. 122:103.
8. Corti, A. et al. (2020) Free Radic. Biol. Med.160:807.
9. Sian, J. et al. (1994) Ann. Neurol. 36:356.
10. Whitfield, J. B. (2001) Crit. Rev. Clin. Lab Sci. 38:263.
11. Lyu, S.C. et al. (2021) Cancer Manag. Res. 13:4887.
12. Carr, B.I. et al. (2021) J. Transl. Sci. 7:10.15761.
13. Sayan, M. et al. (2021) Wiad. Lek. 74: 1804.
14. Jia, Y. et al. (2021) J. Oncol. 9120265.

Publications for GGT1 (10977-GT)(1)

Publication using 10977-GT

• Sivakumar, A;Phuengkham, H;Rajesh, H;Mac, QD;Rogers, LC;Silva Trenkle, AD;Bawage, SS;Hincapie, R;Li, Z;Vainikos, S;Lee, I;Xue, M;Qiu, P;Finn, MG;Kwong, GA; AND-gated protease-activated nanosensors for programmable detection of anti-tumour immunity Nature nanotechnology 2025-01-03 [PMID: 39753733] (Bioassay, Mouse, N/A)

Bioinformatics

• Uniprot:
  • P19440.2()