• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to oxidize 3-hydroxy kynurenine. The specific activity is > 75 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Spodoptera frugiperda,     Sf 21 (baculovirus)-derived Met1-Asn465, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met1

• Predicted Molecular Mass

  54 kDa

• SDS-PAGE

  51 kDa, reducing conditions

Assay Procedure

Materials

• 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:

     *Adjusted for Substrate Blank
     **Derived using calibration standard 3-hydroxyanthranilinic acid (Sigma, Catalog # H9391).

Per Well:

• rhKYNU: 0.1 µg

• Substrate: 100 µM

Background: Kynureninase

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).

• References:

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.

• Entrez Gene IDs:

  8942 (Human); 70789 (Mouse); 116682 (Rat)

• Alternate Names:

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