Recombinant Human BCAT2 Protein, CF

Recombinant Human BCAT2 (Catalog # 9537-BA) is measured byits ability to convert leucine and alpha-ketoglutarate to alpha-ketoisocaproateand glutamate.

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

Recombinant Human BCAT2 Protein, CF Summary

• Details of Functionality: Measured by its ability to convert leucine and alpha-ketoglutarate to alpha-ketoisocaproate and glutamate. The specific activity is >3,500 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human BCAT2 protein
  Ala28-Val392
  with an N-terminal Met and 6-His tag
• Accession #: O15382-1
• N-terminal Sequence: Met
• 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: 42 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: 37-42 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, NaCl, DTT and Glycerol.
• 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 BCAT2 (rhBCAT) (Catalog # 9537-BA)
  • Recombinant Human NQO-1 (rhNQO-1) (Catalog # 7567-DH)
  • Glutamate dehydrogenase (GIDH) (Sigma, Catalog # G7882), 200 U/mL sock 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)
  • alpha -Ketoglutaric Acid (Sigma, Catalog # K2010), 1 M stock in deionized water
  • L-Leucine (EMD Biosciences, Inc., Catalog # 4330), 100 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 rhBCAT2 to 0.1 µg/mL in Assay Buffer.
  2. Dilute alpha -Ketoglutaric Acid to 100 mM in Assay Buffer.
  3. Prepare Substrate Mixture containing 100 U/mL GIDH, 2 mM beta -NAD, 40 µM Resazurin, 1 mM alpha -Ketoglutaric Acid, 4 mM L-Leucine, and 4 µg/mL rhNQO-1 in Assay Buffer.
  4. Load 50 µL of 0.1 µg/mL rhBCAT2 in a plate, 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.
  5. Read at excitation and emission wavelengths of 540 nm and 585 nm (top read), respectively, in kinetic mode for 8 minutes with a three minute lag time 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:
  • rhBCAT2: 0.005 µg
  • GIDH: 5 U
  • beta -NAD: 1 mM
  • rhNQO-1: 0.2 µg
  • Resazurin: 0.02 mM
  • alpha -Ketoglutaric Acid: 0.5 mM
  • L-Leucine: 2 mM

Notes

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

Alternate Names for Recombinant Human BCAT2 Protein, CF

• BCAM
• BCAT(m)
• BCAT2
• BCATM
• BCT2
• BCT2EC 2.6.1.42
• branched chain amino-acid transaminase 2, mitochondrial
• branched chain aminotransferase 2, mitochondrial
• branched-chain-amino-acid aminotransferase, mitochondrial
• Eca40
• Placental protein 18
• PP18

Background

Branched-chain-amino-acid aminotransferases (BCATs) are enzymes that catalyze the first reaction in the catabolism of the essential branched-chain amino acids leucine, isoleucine, and valine to their respective keto-acids while concurrently producing glutamate. BCATs belong to the class-IV pyridoxal-phosphate-dependent (PLP-dependent) aminotransferase family of enzymes (1). There are two BCAT isozymes in humans and mammals, a mitochondrial form known as BCATm or BCAT2 and a cytosolic form known as BCATc or BCAT1 that share 55% sequence identity. In humans and rodents, BCATc is almost exclusively present in the nervous system (2,3) while BCATm is constitutively expressed in most tissues and is generally thought to be important in body nitrogen metabolism (1,2). The 41 kDa human BCATm exists as an active homodimer and has a unique CXXC active site near the dimerization domain (4). Knockouts display decreased adiposity and obesity through alterations of leucine-dependent mTOR signaling making it a potential therapeutic target for obesity (5). BCATm can be regulated by oxidative stress, interfering with its ability to interact with protein disulfide isomerase (6). BCATm overexpression in the brain is detectable in Alzheimers disease and dementia (7,8) suggesting it plays an important role in glutamate toxicity, a key pathogenic feature of these diseases.

1. Hutson, S. (2001) Prog. Nucleic. Acid Res. Mol. Biol. 70:175.
2. Suryawan, A. et al. (1998) Am. J. Clin. Nutr. 68:72.
3. Hall, T.R. et al. (1993) J. Biol. Chem. 268:3092.
4. Yennawar, N. H. (2006) J. Biol. Chem. 281:39660. (BCATm)
5. She, P. et al. (2007) Cell Metab. 6:181.
6. El Hindy, M. et al. (2014) Antioxid. Redox Signal 20:2497.
7. Ashby, E.L. et al. (2017) Neurochem. Res. 42:306.
8. Hull, J. et al. (2015) J. Alzheimers Dis. 45:891.

Publications for BCAT2 (9537-BA)(1)

Publication using 9537-BA

• Wadsworth, BJ;Leiwe, M;Minogue, EA;Cunha, PP;Engman, V;Brombach, C;Asvestis, C;Sah-Teli, SK;Marklund, E;Koivunen, P;Ruas, JL;Rundqvist, H;Lanner, JT;Johnson, RS; A 2-hydroxybutyrate-mediated feedback loop regulates muscular fatigue eLife 2024-09-03 [PMID: 39226092] (Bioassay, Bovine, Mouse)

Bioinformatics

• Uniprot:
  • O15382-1()