Recombinant Human Kynurenine 3-Monooxygenase/KMO Protein, CF

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

Recombinant Human Kynurenine 3-Monooxygenase/KMO Protein, CF Summary

• Details of Functionality: Measured by the consumption of NADPH by hydroxylation of L-kynurenine to 3-hydroxy-kynurenine. The specific activity is >290 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Kynurenine 3-Monooxygenase/KMO protein
  Asp2-Leu441, with an N-terminal Met and 6-His tag
• Accession #: O15229
• N-terminal Sequence: Inconclusive results, intact N-terminus verified by anti-poly-His Western
• Protein/Peptide Type: Recombinant Enzymes
• Gene: KMO
• Purity: >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 51 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: 40-45 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Brij-35.
• Purity: >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM Sodium Phosphate, 0.1% (w/v) Brij-35, pH 7.5
  • Recombinant Human Kynurenine 3-Monooxygenase/KMO (rhKMO) (Catalog # 8050-KM)
  • beta -Nicotinamide adenine dinucleotide phosphate reduced, tetrasodium salt ( beta -NADPH) (Sigma, Catalog # N7505), 10 mM stock in deionized water
  • L-Kynurenine (Tocris, (Catalog # 4393), 40 mM stock in 80 mM HEPES, pH 8.0
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhKMO to 20 ng/μL in Assay Buffer.
  2. Prepare a Reaction Mixture containing 400 μM beta -NADPH and 600 μM L-Kynurenine in Assay Buffer.
  3. In a plate, load 50 μL of 20 ng/μL rhKMO, and start the reaction by adding 50 μL of Reaction Mixture.
  4. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Reaction Mixture.
  5. Read at an absorbance of 340 nm in kinetic mode for 10 minutes.
  6. Calculate specific activity:

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

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 6270 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rhKMO: 1 μg
  • beta -NADPH: 200 μM
  • L-Kynurenine: 300 μM

Notes

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

Alternate Names for Recombinant Human Kynurenine 3-Monooxygenase/KMO Protein, CF

• KMO
• kynurenine 3-monooxygenase (kynurenine 3-hydroxylase)
• Kynurenine 3Monooxygenase
• Kynurenine 3-Monooxygenase

Background

Kynurenine 3-monooxygenase (KMO), also known as kynurenine 3-hydroxylase, is a part of the kynurenine pathway of tryptophan degradation (1). KMO catalyzes the NADPH- and flavin adenine dinucleotide (FAD)-dependent 3-hydroxylation of kynurenine to 3-hydroxykynurenine (3-HK). 3-HK is neurotoxic via the generation of hydrogen peroxide (2) and through the excitotoxic effects of its downstream metabolite quinolinic acid (3). The levels of 3-HK and quinolinic acid are increased in the brain with Alzheimer's disease and Huntington's disease (1). Inhibition of KMO was shown to reverse cognitive and motor deficits in mouse models of those diseases via an increase in neuroprotective kynurenic acid (4). KMO is found in the mitochondrial outer membrane of microglial cells in the brain and dendritic cells and macrophages in the periphery (1). This recombinant human KMO was expressed as a C-terminally truncated protein.

1. Schwarcz, R. et al. (2012) Nat. Rev. Neurosci. 13:465.
2. Okuda, S. et al. (1996) Proc. Natl. Acad. Sci. 93:12553.
3. Stone, T.W. and M.N. Perkins. (1981) Eur. J. Pharmacol. 72:411.
4. Zwilling, D. et al. (2011) Cell 145:863.

Publications for Kynurenine 3-Monooxygenase/KMO (8050-KM)(2)

Publications using 8050-KM

• RB Nahomi, MH Nam, J Rankenberg, S Rakete, JA Houck, GC Johnson, DL Stankowska, MB Pantcheva, PS MacLean, RH Nagaraj Kynurenic Acid Protects Against Ischemia/Reperfusion-Induced Retinal Ganglion Cell Death in Mice Int J Mol Sci, 2020-03-05;21(5):. 2020-03-05 [PMID: 32151061] (Bioassay, Mouse)
• KR Jacobs, GJ Guillemin, DB Lovejoy Development of a Rapid Fluorescence-Based High-Throughput Screening Assay to Identify Novel Kynurenine 3-Monooxygenase Inhibitor Scaffolds SLAS Discov, 2018-02-08;0(0):2472555218757. 2018-02-08 [PMID: 29420107] (Bioassay, Human)

Bioinformatics

• Gene Symbol: KMO
• Uniprot:
  • O15229()