详细说明
- Purity>80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
- Endotoxin Level<1.0 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured 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. See Activity Assay Protocol on .
- SourceSpodoptera frugiperda, Sf 21 (baculovirus)-derived Asp2-Leu441, with an N-terminal Met and 6-His tag
- Accession #
- N-terminal Sequence
AnalysisInconclusive results, intact N-terminus verified by anti-poly-His Western - Predicted Molecular Mass51 kDa
- SDS-PAGE40-45 kDa, reducing conditions
| 8050-KM | | |
| Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Brij-35. | ||
| Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. | ||
| Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
Materials
- 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 # ), 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
- Dilute rhKMO to 20 ng/μL in Assay Buffer.
- Prepare a Reaction Mixture containing 400 μM beta -NADPH and 600 μM L-Kynurenine in Assay Buffer.
- In a plate, load 50 μL of 20 ng/μL rhKMO, and start the reaction by adding 50 μL of Reaction Mixture.
- Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Reaction Mixture.
- Read at an absorbance of 340 nm in kinetic mode for 10 minutes.
- Calculate specific activity:
| Specific Activity (pmol/min/µg) = | Adjusted Vmax* (OD/min) x -1 x well volume (L) x 1012 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
Background: Kynurenine 3-Monooxygenase/KMO
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.
- References:
- Schwarcz, R. et al. (2012) Nat. Rev. Neurosci. 13:465.
- Okuda, S. et al. (1996) Proc. Natl. Acad. Sci. 93:12553.
- Stone, T.W. and M.N. Perkins. (1981) Eur. J. Pharmacol. 72:411.
- Zwilling, D. et al. (2011) Cell 145:863.
- Entrez Gene IDs:8564 (Human); 98256 (Mouse); 59113 (Rat)
- Alternate Names:KMO; kynurenine 3-monooxygenase (kynurenine 3-hydroxylase); Kynurenine 3Monooxygenase; Kynurenine 3-Monooxygenase
400-6226-992