Recombinant Human GOT1 His-tag Protein, CF

Recombinant Human GOT1 His-tag Protein (Catalog # 11696-G1) is measured by its ability to produce L-glutamate from L-aspartate in a coupled reaction.

2 μg/lane of Recombinant Human GOT1 His-tag Protein (Catalog # 11696-G1) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 38-42 kDa under reducing conditions.

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

Recombinant Human GOT1 His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to produce L-glutamate from L-aspartate in a coupled reaction.
  The specific activity is >15,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human GOT1 protein
  Ala2-Gln413, with an N-terminal Met and 6-His tag
• Accession #: P17174.3
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 47 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: 38-42 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.
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, pH 7.5
  • Recombinant Human GOT1 His-tag (rhGOT1) (Catalog # 11696-G1)
  • Recombinant Human MDH1 His-tag (rhMDH1) (Catalog # 11632-MH)
  • Substrate: L-Aspartic Acid, 20 mM stock in deionized water
  • beta -Nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH), 20 mM stock in 0.1 M Sodium Borate, pH 9.0
  • alpha -Ketoglutaric Acid, 1 M stock in deionized water
  • Pyridoxal 5-phosphate, 100 mM stock in 1 M HEPES, pH 8.0
  • Clear 96-well Plate
  • Plate Reader with Absorbance Read Capability
  1. Prepare a Reaction Mixture containing 1 mM NADH, 1 mM alpha -Ketoglutaric Acid, 20 µg/mL rhMDH1, and 40 µM Pyridoxal 5-phosphate in Assay Buffer.
  2. Dilute Substrate to 6 mM in Assay Buffer.
  3. Dilute rhGOT1 to 0.8 µg/mL in Assay Buffer.
  4. In a clear well plate, combine 25 µL of 0.8 µg/mL rhGOT1 and 25 µL of Reaction Mixture. Include a Control containing 25 µL of Assay Buffer and 25 µL of Reaction Mixture.
  5. Start the reaction by adding 50 µL of 6 mM Substrate to all wells.
  6. Read plate in kinetic mode for 6 minutes with a 1 minute lag at an absorbance of 340 nm.
  7. Calculate specific activity:

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

  
  
  *Adjusted for Control
  **Using the extinction coefficient 6220 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD
  Per Well:
  • rhGOT1: 0.020 µg
  • rhMDH1: 5 µg/mL
  • Substrate: 3 mM
  • NADH: 250 µM
  • alpha -Ketoglutaric Acid: 250 µM
  • Pyridoxal 5-phosphate: 10 µM

Notes

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

Alternate Names for Recombinant Human GOT1 His-tag Protein, CF

• aspartate aminotransferase, cytoplasmic
• AST1
• ASTQTL1
• CASPAT
• CCAT
• Cysteine aminotransferase, cytoplasmic
• Cysteine transaminase, cytoplasmic
• EC 2.6.1.1
• EC 2.6.1.3
• GIG18
• Glutamate oxaloacetate transaminase 1
• glutamic-oxaloacetic transaminase 1, soluble (aspartate aminotransferase 1)
• GOT1
• growth-inhibiting protein 18
• SGOT
• Transaminase A

Background

Glutamate oxaloacetate transaminase 1 (GOT1), also known as cytoplasmic Aspartate aminotransferase (ATTC) belongs to the class-I pyridoxal-phosphate-dependent aminotransferase family (1). GOT1 is broadly expressed across tissues, organs, and cells (2, 3). GOT1 has an isozyme, GOT2, with 48% similarity (4) that differs in cellular localization. While the GOT2 isozyme is the mitochondrial form, GOT1 is the cytosolic form. GOT1 is an active homodimer of monomers with a small domain that contains the N- and C-terminus and a large domain that contains the core structure and dimerization surface. Each monomer contains an active site and a pyridoxal 5'-phosphate (PLP) cofactor (5). GOT1 is involved in biosynthesis of L-glutamate from L-aspartate or L-cysteine and functions as a critical enzyme in the malate-aspartate shuttle where it plays a role in the regulation of metabolic activity in human cells and also in cancer cells where it is required to maintain enhanced glycolysis to support proliferation (1, 3). GOT1 is overexpressed in numerous cancers including pancreatic, colon, breast, prostate, and leukemina and correlated with poor prognosis (6-11) giving it value as a diagnostic marker (1,3).  In addition, GOT-1 expression has been reported as an indicator for liver dysfunction, cardiovascular disease, and diabetes (12). Given the broad implications of GOT1 involvement in disease and as a cancer metabolic target of interest, there is significant interest in GOT-1 as a therapeutic target (3, 8,13,14). 

1. Peng, H. et al. (2024) Front. Oncol. 14:1519046. 
2. Ladue, J.S. et al. (1954) Science. 120:497.
3. Song, Z. et al. (2022) Eur. J. Pharmacol. 917:174754. 
4. Doyle, J.M. et al. (1990) Biochem. J. 270:651. 
5. Malashkevich, V.N. et al. (1995) J. Mol. Biol. 247:111. 
6. Feld, F.M. et al. (2015) Oncotarget 6:4516.
7. Yang, Y. (2016) Biochem. Biophys. Res. Commun. 473:1295.
8. Hong, C. et al. (2017) Cancer Chemother. Pharmacol. 79:835.
9. Cheng, Z. et al. (2020) Front. Oncol. 10:379.
10. Singh, A.N. et al. (2020) Front. Oncol. 10:493.
11. Siwo, G.H. et al. (2024) preprint. doi: 10.1101/2024.03.01.582939.
12. Monami, M. et al. (2008) Metabolism 57:387.
13. Holt, M.C. et al. (2018) Biochemistry 57:6604. 
14. Wang, Q. et al. (2019) Bioorg. Chem. 93:103315.

Bioinformatics

• Uniprot:
  • P17174.3()