详细说明
- Purity>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
- Endotoxin Level<1.0 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured by its ability to hydrolyze thymidine 5'-monophosphate p-nitrophenyl ester. The specific activity is >22,000 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .
- SourceHuman embryonic kidney cell, HEK293-derived Tyr19-Ala410, with a C-terminal 6-His tag
- Accession #
- N-terminal Sequence
AnalysisTyr19 - Predicted Molecular Mass45 kDa
- SDS-PAGE56-74 kDa, reducing conditions
| 8996-EN | | |
| Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. | ||
| Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. | ||
| Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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- Assay Buffer: 50 mM Tris, pH 7.5
- Recombinant Mouse ENPP-4 (rmENPP-4) (Catalog # 8996-EN)
- Substrate: Thymidine 5’-monophosphate p-nitrophenyl ester (Sigma, Catalog # T4510), 100 mM stock in deionized water
- NaOH, 0.2 M in deionized water
- 96-well Clear Plate (Catalog # )
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rmENPP-4 to 0.1 ng/µL in Assay Buffer.
- Dilute Substrate to 10 mM in Assay Buffer.
- Load 50 µL of 0.1 ng/µL rmENPP-4 in a clear strip well plate, and start the reaction by adding 50 µL of 10 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL 10 mM Substrate.
- Incubate sealed plate at room temperature for 30 minutes.
- Stop reactions by adding 100 µL of 0.2 M NaOH to all wells, including Substrate Blank wells.
- Read at 410 nm (absorbance) in endpoint mode.
- Calculate specific activity:
| Specific Activity (pmol/min/µg) = | Adjusted Abs* (OD) x Conversion Factor** (pmol/OD) |
| Incubation time (min) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard p-Nitrophenol (Sigma, Catalog # 241326). Per Reaction:- rmENPP-4: 0.005 µg
- Substrate: 5 mM
Ectonucleotide pyrophosphatase/phosphodiesterase 4 (ENPP-4 or NPP4) belongs to a group of ecto-enzymes which regulate the availability of extracellular nucleotides (1). This enzyme family forms a subgroup of a larger family that also includes arylsulfatases, phosphopentomutases, 2,3-bisphosphoglycerate-independent phosphoglycerate mutases (iPGM), and alkaline phosphatases (2). Mature mouse ENPP-4 consists of a 392 amino acid (aa) ectodomain that contains the catalytic domain with a zinc-coordinated substrate binding pocket, a 21 aa transmembrane segment, and a 25 aa cytoplasmic tail (3). It shares 86% and 90% aa sequence identity with human and rat ENPP-4, respectively. Alternative splicing generates a short isoform with a 32 aa deletion in the phosphodiesterase domain. ENPP-4 hydrolyzes phosphodiester bonds in nucleotides with a preference for adenine nucleotides (3). It cleaves the diadenosine compounds Ap3A and Ap4a which are released from the dense granules of thrombin-activated platelets (3, 4). These reactions generate AMP and ADP from Ap3A cleavage, and AMP and ATP from Ap4A cleavage (4). ENPP-4 is expressed on the surface of vascular endothelial cells where its activity prolongs platelet aggretation and contributes to thrombus formation (4).
- References:
- Zimmermann, H. et al. (2012) Purinergic Signal. 8:437.
- Gijsbers, R. et al. (2001) J. Biol. Chem. 276:1361.
- Albright, R.A. et al. (2014) J. Biol. Chem. 289:3294.
- Albright, R.A. et al. (2012) Blood 120:4432.
- Long Name:Ectonucleotide Pyrophosphatase/Phosphodiesterase 4
- Entrez Gene IDs:22875 (Human); 224794 (Mouse); 301261 (Rat)
- Alternate Names:AP3Aase; EC 3.1; ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative); E-NPP 4; ENPP4; ENPP-4; KIAA0879ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function); NPP-4; NPP4ectonucleotide pyrophosphatase/phosphodiesterase family member 4
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