Recombinant Human IMP Dehydrogenase 2/IMPDH2 Protein, CF

1 μg/lane of Recombinant Human Inosine 5'-Monophosphate Dehydrogenase 2/IMPDH2 was resolved with SDS-PAGE underreducing (R) and non-reducing (NR) conditions and visualized by silverstaining,showing a band at 57 kDa.

Recombinant Human IMPDH2 (Catalog # 8349-DH) is measured byits ability to convert the substrate inosine-5'-phosphate (IMP) toxanthosine-5'-phosphate (XMP). The activity (orange) is approximately 3.5-foldgreater than the competitor's IMPDH2 (green).

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

Recombinant Human IMP Dehydrogenase 2/IMPDH2 Protein, CF Summary

• Details of Functionality: Measured by its ability to convert the substrate inosine-5'-phosphate (IMP) to xanthosine-5'-phosphate (XMP). The specific activity is >30 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human IMP Dehydrogenase 2/IMPDH2 protein
  Met1-Phe514, with a C-terminal 10-His tag
• Accession #: P12268
• N-terminal Sequence: Met1 predicted
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >90%, 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: 57 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: 52-61 kDa, 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, DTT and Glycerol.
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 8.0
  • Recombinant Human Inosine 5'-Monophosphate Dehydrogenase 2/IMPDH2 (rhIMPDH2) (Catalog # 8349-DH)
  • Nicotinamide adenine dinucleotide sodium salt ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in deionized water
  • Inosine 5'-monophosphate (IMP) (Sigma, Catalog # I4625), 100 mM stock in deionized water.
  • UV plate (Costar, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhIMPDH2 to 20 μg/mL in Assay Buffer.
  2. Create Substrate Mixture containing 500 μM IMP and 1 mM beta -NAD in Assay Buffer.
  3. Load 50 μL of 20 μg/mL rhIMPDH-2 into a plate, and start the reaction by adding 50 μL of Substrate Mixture. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of Substrate Mixture.
  4. Read plate at a wavelength of 339 nm (bottom read) in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) 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 6220 M^-1cm^-1.
  ***Using the path correction 0.320 cm.
  Note: the output of many spectrophotometers is in mOD.

  Per Well:
  • rhIMPDH-2: 1.0 μg
  • beta -NAD: 500 μM
  • IMP: 250 μM

Notes

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

Alternate Names for Recombinant Human IMP Dehydrogenase 2/IMPDH2 Protein, CF

• EC 1.1.1.205
• IMDH2
• IMP (inosine 5'-monophosphate) dehydrogenase 2
• IMP (inosine monophosphate) dehydrogenase 2
• IMP Dehydrogenase 2
• IMP Oxireductase 2
• IMPD 2
• IMPD2
• IMPD2IMP oxireductase 2
• IMPDH 2
• IMPDH2
• IMPDH-II
• inosine 5' phosphate dehydrogenase 2
• inosine monophosphate dehydrogenase type II
• inosine-5'-monophosphate dehydrogenase 2

Background

IMPDH2 (inosine monophosphate dehydrogenase) is one of two cytosolic and nuclear enzyme isoforms that play a central role in guanine nucleotide metabolism. The isoforms are 84% identical but distinctly regulated. While IMPDH1 is generally constitutively expressed, IMPDH2 is inducible during proliferation and transformation (1, 2). Both isoforms form a homotetramer of approximately 55 kDa monomers containing a catalytic barrel domain and a subdomain with two cystathione beta-synthase domains which mediate RNA and DNA binding (1, 3). Both enzymes catalyze the NAD-dependent conversion of inosine monophosphate (IMP) to hypoxanthine monophosphate (XMP) which is a precursor for the synthesis of GMP, guanosine, and guanine. These compounds are critical for DNA synthesis and cell proliferation (4, 5) which explains the importance of IMPDH in cancer and viral infection (6-9). Although IMPDH1 and IMPDH2 are known to be inhibited by the immunosuppressant drug mycophenolic acid (MPA), much research has targeted discovery of additional and selective inhibitors for IMPDH isoforms given they are targets for several major therapeutic areas (1, 9-13). Human IMPDH2 shares 99% amino acid sequence identity with mouse IMPDH2.

1. Hedstrom, L. (2009) Chem. Rev. 109:2903.
2. Thomas, E.C. et al. (2012) PLoS One 12:e51096.
3. McLean, J.E. et al. (2004) Biochem. J. 379:243.
4. Lane, A.N. and T.W. Fan (2015) Nucleic Acids Res. 43:2466.
5. Jackson, R.C. et al. (1975) Nature 256:331.
6. Zho, J. et al. (2015) Med. Oncol. 32:373.
7. Xu, Y. et al. (2017) Sci. Rep. 7:745.
8. Nair, V. (2007) Antivir. Chem. Chemother. 18:245.
9. Trapero, A. et al. (2018) J. Med. Chem. 61:2806.
10. Barnes, B.J. et al. (2001) Int. J. Cancer 94:275.
11. Cholewinksi, G. et al. (2015) J. Enzyme Inhib. Med. Chem. 30:550.
12. Liao, L.X. et al. (2017) Proc. Natl. Acad. Sci. USA 114:E5986.
13. Cuny, G.D. et al. (2017) Expert Opin. Ther. Pat. 27:677.

Bioinformatics

• Uniprot:
  • P12268()