Recombinant Human Pyridoxal Kinase Protein, CF

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

Recombinant Human Pyridoxal Kinase Protein, CF Summary

• Details of Functionality: Measured by its ability to phosphorylate Pyridoxal Hydrochloride. The specific activity is >85 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Pyridoxal Kinase/PDXK protein
  Glu2-Leu312, with N-terminal Met and 6-His tag
  Accession # O00764
• Accession #: O00764
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Proteins
• Gene: PDXK
• 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: 36 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: 36 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 DTT.
• Purity: >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Universal Kinase Activity Kit (Catalog # EA004)
  • 10X Assay Buffer (supplied in kit): 250 mM HEPES, 1.5 M NaCl, 100 mM MgCl₂, 100 mM CaCl₂, pH 7.0
  • Recombinant Human Pyridoxal Kinase/PDXK (rhPDXK) (Catalog # 8658-PK)
  • ATP (supplied in kit): 10 mM
  • Pyridoxal Hydrochloride (Sigma, Catalog # P6155), 50 mM in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare 1X Assay Buffer by diluting 10X stock with deionized water.
  2. Dilute 1 mM Phosphate Standard supplied in the Universal Kinase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  3. Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  4. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
  5. Prepare a reaction mixture containing 0.4 mM ATP and 2 mM Pyridoxal Hydrochloride in 1X Assay Buffer.
  6. Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in 1X Assay Buffer.
  7. Dilute rhPDXK to 133 µg/mL in 1X Assay Buffer.
  8. Load 15 µL of the 133 µg/mL rhPDXK into empty wells of the same plate as the curve. Include a control containing 15 µL of 1X Assay Buffer.
  9. Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to wells containing enzyme and control, excluding the standard curve.
  10. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  11. Seal plate and incubate at room temperature for 10 minutes.
  12. Add 30 µL of the Malachite Green Reagent A to all wells.
  13. Add 100 µL of deionized water to all wells. Mix briefly.
  14. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  15. Read plate at 620 nm (absorbance) in endpoint mode.
  16. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Phosphate released* (nmol) x (1000 pmol/nmol)) / (Incubation time (min) x amount of enzyme (µg) x coupling rate**)

  
  *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control
  **The coupling rate is 0.475 under these conditions.

  Per Well:
  • rhPDXK: 2.0 µg
  • Coupling Phosphatase 4: 0.1 µg
  • ATP: 0.2 mM
  • Pyridoxal Hydrochloride: 1 mM

Notes

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

Alternate Names for Recombinant Human Pyridoxal Kinase Protein, CF

• C21orf124
• C21orf97
• chromosome 21 open reading frame 124
• DKFZp566A071
• EC 2.7.1.35
• FLJ21324
• FLJ37311
• HEL-S-1a
• MGC15873
• MGC31754
• MGC52346
• PDXK
• PKH
• PKHchromosome 21 open reading frame 97
• PNK
• PNKhuman pyridoxal kinase, EC 2.7.1.3510FLJ31940
• PRED79
• pyridoxal (pyridoxine, vitamin B6) kinase
• Pyridoxal Kinase
• pyridoxamine kinase
• Pyridoxine kinase
• Vitamin B6 Kinase

Background

Pyridoxal Kinase (PDXK) phosphorylates vitamin B6, a step required for the conversion of vitamin B6 to pyridoxal-5-phosphate (PLP), an important cofactor in intermediary metabolism (1). PLP is involved in many aspects of macronutrient metabolism, neurotransmitter synthesis, histamine synthesis, as well as hemoglobin synthesis, function, and gene expression (2). PLP generally serves as a co-enzyme for many reactions and can help facilitate decarboxylation, transamination, racemization, elimination, replacement and beta-group inter-conversion reactions. In particular, PLP also is needed for the enzymatic reaction governing the release of glucose from glycogen. Overall, there are over 160 PLP-dependent enzymes, corresponding to ~4% of all known enzymatic activities (3). The classic clinical syndrome for B6 deficiency is a seborrhoeic dermatitis-like eruption, atrophic glossitis with ulceration, angular cheilitis, conjunctivitis, intertrigo, accompanied by neurologic symptoms of somnolence, confusion, and neuropathy (4).The neurotoxic effects of ophylline and ginkgotoxin are caused by their inhibitory activity against human PDXK (3, 5). Genetic variations of PDXK are associated with Parkinson disease, since PLP is involved in the biosynthesis of dopamine, a neurotransmitter linked to the disease characteristics such as motor and movement disorders (6). PDXK is also a potential drug target in the African trypanosomiasis (7). PDXK is a cytoplasmic enzyme and probably acts as a homodimer in vivo (8). The enzymatic activity of recombinant human CHKB is measured using a phosphatase-coupled method (9).

1. Hanna, M.C. et al. (1997) J. Biol. Chem. 272:10756.
2. Percudani R. and A. Peracchi (2003). EMBO Rep. 4: 850.
3. Gandhi, AK et al. (2012) PLoS ONE. 7:e40954.
4. Lichtstein, H.C. et al. (1945) J. Biol. Chem. 161:311.
5. Kastner, U. et al. (2007) FEBS J. 274:1036.
6. Vilarino-Guell, C. et al. (2010) Ann. Neurol. 67:409.
7. Jones, D.C. et al. (2012) Mol. Microbiol. 86:51.
8. Li , M.H. et al. (2002) J. Biol. Chem. 277:46385.
9. Wu, Z.L. (2011) PLoS ONE 6:e23172.

Publications for Pyridoxal Kinase/PDXK (8658-PK)(1)

Publication using 8658-PK

• Franco, CN;Seabrook, LJ;Nguyen, ST;Yang, Y;Campos, M;Fan, Q;Cicchetto, AC;Kong, M;Christofk, HR;Albrecht, LV; Vitamin B6 is governed by the local compartmentalization of metabolic enzymes during growth Science advances 2023-09-08 [PMID: 37682999] (Bioassay, N/A)

Bioinformatics

• Gene Symbol: PDXK
• Uniprot:
  • O00764()