Recombinant Human NM23-H1 His-tag Protein, CF

Recombinant Human NM23-H1 His-tag (Catalog # 10495-NM) is measured by its ability to convert thymidine diphosphate and adenosine triphosphate to thymidine triphosphate and adenosine diphosphate in a coupled assay.

2 μg/lane of Recombinant Human NM23-H1 His-tag Protein (Catalog # 10495-NM) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 18-20 kDa.

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

Recombinant Human NM23-H1 His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to convert thymidine diphosphate and adenosine triphosphate to thymidine triphosphate and adenosine diphosphate in a coupled assay.
  The specific activity is >50,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human NM23-H1 protein
  Ala2-Glu152
  with a C-terminal 6-His tag
• Accession #: P15531.1
• N-terminal Sequence: Ala2
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 18 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: 18-20 kDa, under 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 Tris, NaCl and TCEP.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM MgCl₂, 100 mM KCl, pH 7.5
  • Recombinant Human NM23-H1 His-tag (rhNM23-H1) (Catalog # 10495-NM)
  • Substrate: Thymidine 5'-Diphosphate (TDP) (Sigma, Catalog # T9375), 40 mM stock in 10 mM Sodium Borate, pH 9.0
  • Recombinant Human PKM2 (rhPKM2) (Catalog # 7244-PK)
  • Recombinant Human Lactate Dehydrogenase A/LDHA (rhLDHA) (Catalog # 9158-HA)
  • Adenosine 5'-triphosphate (ATP) (Sigma, Catalog # A7699), 400 mM stock in deionized water
  • beta -Nicotinamide adenine dinucleotide, reduced disodium salt hydrate ( beta -NADH) (Sigma, Catalog # N8129), 20 mM stock in 0.1 M Sodium Borate, pH 9.0
  • Phospho(enol)pyruvic acid (PEP) (Sigma, Catalog # P0564), 50 mM stock in deionized water
  • 96-well clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare Reaction Mixture containing 20 µg/mL rhPKM2, 5 µg/mL rhLDHA, 4 mM ATP, 400 µM beta -NADH, 2 mM PEP and 2 mM TDP in Assay Buffer.
  2. Incubate Reaction Mixture at room temperature for 5 minutes.
  3. Dilute rhNM23-H1 to 0.01 ng/µL in Assay Buffer.
  4. In a plate, load 50 µL of 0.01 ng/µL rhNM23-H1 and start the reaction by adding 50 µL of Reaction Mixture. Include a Control containing 50 µL of Assay Buffer and 50 µL of Reaction Mixture.
  5. Read at an absorbance of 340 nm in kinetic mode for 5 minutes.
  6. Calculate specific activity:

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

  *Adjusted for Control
  **Using the extinction coefficient 6220 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD

  Per Well:
  • rhNM23-H1: 0.0005 µg
  • TDP: 1 mM
  • ATP: 2 mM
  • beta -NADH: 200 µM
  • PEP: 1 mM
  • rhPKM2: 1 µg
  • rhLDHA: 0.25 µg

Notes

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

Alternate Names for Recombinant Human NM23-H1 His-tag Protein, CF

• EC 2.7.4.6
• GAAD
• Granzyme A-activated DNase
• Metastasis inhibition factor nm23
• NB
• NBS
• NDK A
• NDKA
• NDP kinase A
• NDPKA
• NDPK-A
• NM23A
• NM23AWD
• NM23H1
• NM23-H1
• NME1
• non-metastatic cells 1, protein (NM23A) expressed in
• nucleoside diphosphate kinase A
• Tumor metastatic process-associated protein

Background

Non-metastatic Protein 23 Homolog 1 (NM23-H1), also known as Nucleoside diphosphate kinase A (NDPK-A), is a group 1 member of the NDPK family encoded by the NME1 (non metastatic cell) gene (1). NDPKs are well-known as housekeeping genes that maintain the balance of nucleoside triphosphate levels by transferring a phosphate group from a nucleoside triphosphate to nucleoside diphosphate. NM23-H1 is a cytosolic protein that forms active hexamers composed of a trimer of dimers (2,3). The active hexamer contains a "head" region with substrate binding and catalytic activity as well as a Kpn loop and C-terminal extensions that stabilize the structure (2,3). NM23-H1 (NDPK-A) shares significant homology with NM23-H2 (NDPK-B), with 88% identity (3). In spite of significant homology and catalytic function between these isoforms, the differing amino acids on the lateral surface of the hexamers lead to specific interactions with other proteins and ultimately result in diverging cellular distribution and function in cells (1). NM23-H1 was the first reported metastasis suppressor gene (4) and plays a role in cell adhesion, migration and motility, and signaling and proteolysis (5-7). Suppression activity has been demonstrated in many cancers although the mechanism involved is uncertain (1,7). NM23-H2 has been reported to have several functions: enzymatic function as a protein histidine kinase with ATP-citrate lyase and annexin A1 as direct targets (5), exonuclease activity (8), NDPK activity (1), and also forms a complex with the cystic fibrosis transmembrane conductance regulator (CFTR) (9) in regulation of chloride channels, other signaling pathways, and gene regulation (1,10,11).

1. Boissan, M. et al. (2018) Lab. Invest. 98:164.
2. Janin, J. et al. (2000) J. Bioenerg. Biomembr. 32:215.
3. Webb, P.A. et al. (1995) J. Mol. Biol. 251:574.
4. Steeg, P.S. et al. (1988) J. Natl. Cancer Inst. 80:200.
5. Attwood, P.V. et al. (2018) Lab. Invest. 98:283.
6. Khan, I. et al. (2019) Cancer Res. 79:4689.
7. Matyasi, B. et al. (2020) Pathol. Oncol. Res. 26:49.
8. Fan, Z. et al. (2003) Cell 112:659.
9. Borthwick, L.A. et al. (2016) PloS One. 11:e0149097.
10. Ma, W. et al. (2008) Biochem. Biophys. Res. Commun. 371:425.
11. Zheng, S. et al. (2019) FEBS Lett. 593:80.
    

Bioinformatics

• Uniprot:
  • P15531.1()