Recombinant Human ENPP-1 Protein, CF

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

Recombinant Human ENPP-1 Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze thymidine 5'-monophosphate p-nitrophenyl ester. The specific activity is >35,000 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human ENPP-1 protein
  

  His	ENPP-2 Asp49-Trp144 Accession # Q13822	ENPP-1 Val191-Leu591 Accession # P22413	ENPP-2 Asn532-Ile863 Accession # Q13822
  N-terminus			C-terminus

• Accession #: P22413 (ENPP-1) & Q13822 (ENPP-2)
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: ENPP1
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 96 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: 100-135 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 and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 250 mM NaCl, pH 9.5
  • Recombinant Human ENPP-1 (rhENPP-1) (Catalog # 6136-EN)
  • Substrate: Thymidine 5’-monophosphate p-nitrophenyl ester (Sigma, Catalog # T4510), 100 mM stock in deionized water
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhENPP-1 to 1 ng/µL in Assay Buffer.
  2. Dilute Substrate to 10 mM in Assay Buffer.
  3. Load into a plate 50 µL of 1 ng/µL rhENPP-1, and start the reaction by adding 50 µL of 10 mM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 10 mM Substrate.
  4. Read at 405 nm (absorbance) in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x Conversion Factor** (pmol/OD)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Derived using calibration standard 4-Nitrophenol (Sigma-Aldrich, Catalog # 241326).

  Per Well:
  • rhENPP-1: 0.05 µg
  • Substrate: 5 mM

Notes

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

Alternate Names for Recombinant Human ENPP-1 Protein, CF

• ectonucleotide pyrophosphatase/phosphodiesterase 1
• E-NPP 1
• ENPP1
• ENPP-1
• Ly-41 Antigen
• M6S1
• M6S1NPP1
• Membrane component chromosome 6 surface marker 1
• membrane component, chromosome 6, surface marker 1
• NPP1
• NPPS
• NPPSalkaline phosphodiesterase 1
• PC1
• PC-1
• PCA1
• PCA1ARHR2
• PDNP1
• PDNP1ectonucleotide pyrophosphatase/phosphodiesterase family member 1
• Phosphodiesterase I/nucleotide pyrophosphatase 1
• plasma-cell membrane glycoprotein 1
• Plasma-cell membrane glycoprotein PC-1

Background

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP-1) is a transmembrane glycoprotein that hydrolyzes nucleotides and nucleotide derivatives with the formation of nucleotide-5’-monophosphates. It is inserted into the plasma membrane by an N‑terminal transmembrane domain. Human ENPP-1 has a small N-terminal cytoplasmic domain and a large C-terminal region containing two somatomedin B-like domains, a catalytic domain and a nuclease-like domain in the extracellular space (1). Defects in the ENPP-1 gene cause arterial calcification and bone mineralization abnormalities (2). ENPP-1 polymorphism or overexpression is also associated with obesity, type II diabetes and insulin resistance, which makes modulation of ENPP-1 activity one of the targets to treat insulin resistance and related diseases (1). The recombinant hybrid enzyme recombinant human ENPP-1 consists of N-terminal somatomedin B-like domains of ENPP-2, the central catalytic phosphodiesterase domain of ENPP-1 and the C‑terminal nuclease-like domain of ENPP-2. It has been reported that this hybrid construct generates an enzyme that has higher levels of ENPP-1 activity than the wild type ENPP-1 and is specific for the ENPP-1 substrate (3).

1. Goldfine, I.D. et al. (2010) Endocrine Reviews. 29:62.
2. Hessle, L. et al. (2002) Proc. Natl. Acad. Sci. 99:9445.
3. Anisoara, C. et al. (2004) Biochem. J. 381:71.

Publications for ENPP-1 (6136-EN)(4)

Publications using 6136-EN

• Baird, JR;Alice, AF;Saito, R;Chai, Q;Han, M;Ng, C;Han, S;Fernandez, B;Ledoux, S;Grosse, J;Korman, AJ;Potuznik, M;Rajamanickam, V;Bernard, B;Crittenden, MR;Gough, MJ; A novel small molecule Enpp1 inhibitor improves tumor control following radiation therapy by targeting stromal Enpp1 expression Scientific reports 2024-12-02 [PMID: 39622844] (Bioassay, Human)
• Hong, CS;Menshikova, EV;Whiteside, TL;Jackson, EK; Assessment of ATP Metabolism to Adenosine by Ecto-Nucleotidases Carried by Tumor-Derived Small Extracellular Vesicles Research square 2024-01-24 [PMID: 38343828] (Bioassay, Human)
• Jekle, A;Thatikonda, SK;Jaisinghani, R;Ren, S;Kinkade, A;Stevens, SK;Stoycheva, A;Rajwanshi, VK;Williams, C;Deval, J;Mukherjee, S;Zhang, Q;Chanda, S;Smith, DB;Blatt, LM;Symons, JA;Gonzalvez, F;Beigelman, L; Tumor Regression upon Intratumoral and Subcutaneous Dosing of the STING Agonist ALG-031048 in Mouse Efficacy Models International journal of molecular sciences 2023-11-13 [PMID: 38003463] (Bioassay, Human)
• Madan , Damian, Ferguson , Colin G, Lee , Won Yong, Prestwich , Glenn D, Testa , Charles Non-invasive imaging of tumors by monitoring autotaxin activity using an enzyme-activated near-infrared fluorogenic substrate. PLoS ONE, 2013-11-20;8(11):e79065. 2013-11-20 [PMID: 24278115] (Bioassay)

Bioinformatics

• Gene Symbol: ENPP1
• Uniprot:
  • P22413 (ENPP-1) & Q13822 (ENPP-2)()