Recombinant Human Kynureninase Protein, CF

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

Recombinant Human Kynureninase Protein, CF Summary

• Details of Functionality: Measured by its ability to oxidize 3-hydroxy kynurenine. The specific activity is > 75 pmol/min/µg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Kynureninase protein
  Met1-Asn465, with a C-terminal 10-His tag
• Accession #: NP_003928
• N-terminal Sequence: Met1
• Protein/Peptide Type: Recombinant Enzymes
• Gene: KYNU
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 54 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: 51 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 MES and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 0.05% (w/v) Brij-35, 5 µM Pyridoxal Phosphate (Sigma, Catalog # P9255), pH 8.0
  • Recombinant Human Kynureninase (rhKYNU) (Catalog # 4887-KH)
  • Substrate: 3-hydroxy-DL-kynurenine (3-HK) (Sigma, Cat. # H1771), 10 mM in 50% DMSO, 50% deionized water
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhKYNU to 2 ng/µL in Assay Buffer.
  2. Dilute Substrate to 200 µM in Assay Buffer.
  3. Load in a black well plate 50 µL of 2 ng/µL rhKYNU, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 μL Assay Buffer and 50 µL 200 µM Substrate.
  4. Read at excitation and emission wavelengths of 315 nm and 415 nm (top read), respectively, in kinetic mode for 5 minutes.
  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 3-hydroxyanthranilinic acid (Sigma, Catalog # H9391).

  Per Well:
  • rhKYNU: 0.1 µg
  • Substrate: 100 µM

Notes

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

Alternate Names for Recombinant Human Kynureninase Protein, CF

• EC 3.7.1.3
• KYNU
• kynureninase (L-kynurenine hydrolase)
• Kynureninase
• L-kynurenine hydrolase

Background

Kynureninase is a pyridoxal-5 (-phosphate-dependent enzyme that catalyzes the hydrolytic cleavage of the amino acids L-kynurenine and L-3-hydroxykynurenine to give either anthranilic acid or 3-hydroxyanthranilic acid and alanine (1). The enzyme is a member of the “kynurenine pathway” enzymes, through which the majority of dietary tryptophan is degraded in the liver, and is involved in the de novo biosynthesis of NAD⁺ (2, 3). Kynurenine pathway genes are expressed in immune system cells such as macrophages and microglia. During inflammatory responses, the kynurenine pathway in these cells produces quinolinic acid (QA) and not NAD⁺. QA excites neurons via the activation of NMDA (N-methyl-D-aspartate) receptors resulting in neuronal damage. The tissue-damaging process has been demonstrated in AIDS-related dementia complex, Alzheimer’s, stroke, epilepsy, and Huntington’s disease. Because Kynureninase is one of the key enzymes of QA production, its inhibitors may be useful for the treatment of neurological disorders. The recombinant Kynureninase has been shown to possess specificity for 3-hydroxykynurenine over kynurenine (4, 5).

1. Lima, S. et al. (2007) Biochemistry 46:2735.
2. Botting, N. P. (1995) Chem. Soc. Rev. 24:401.
3. Stone, T. W. (2000) Trends in Pharm. Sci. 21:149.
4. Walsh, H. et al. (2002) Eur. J. Chem. 269:2069.
5. Toma, S. et al. (1997) FEBS Lett. 408:5.

Publications for Kynureninase (4887-KH)(2)

Publications using 4887-KH

• V Morozov, S Lagaye, A Morozov The TZM-bl Reporter Cell Line Expresses Kynureninase That Can Neutralize 2F5-like Antibodies in the HIV-1 Neutralization Assay International Journal of Molecular Sciences, 2022-01-07;23(2):. 2022-01-07 [PMID: 35054825] (Bioassay, Human)
• Barton F Haynes HIV-1 Envelope Mimicry of Host Enzyme Kynureninase Does Not Disrupt Tryptophan Metabolism J. Immunol., 2016-11-14;0(0):. 2016-11-14 [PMID: 27849170] (Bioassay)

Bioinformatics

• Gene Symbol: KYNU
• Uniprot:
  • NP_003928()