Recombinant Mouse VAP-1 Protein, CF
Recombinant Mouse VAP-1 Protein, CF Summary
|Met||10-His tag||GGGSGGGSGGGS||IEGR||Mouse VAP-1/AOC3|
Accession # O70423
|Formulation||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 50 mM HEPES, pH 7.5
- Recombinant Mouse VAP‑1/AOC3 (rmVAP-1) (Catalog # 6107-AO)
- Coupling Agent: Horseradish Peroxidase (HRP) (250-330 U/mg) (Sigma, Catalog # P8375), 250 units/mL stock in 0.1 M Sodium Phosphate, pH 8.0
- Substrate Component 1: Benzylamine (Sigma, Catalog # B5136), 100 mM stock in deionized water
- Substrate Component 2: Amplex Ultra Red (AUR) (Molecular Probes, Catalog # A36006), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rmVAP-1 to 8 ng/µL in Assay Buffer.
- Prepare the Substrate mixture 2 mM Benzylamine, 2 units/mL HRP and 100 µM AUR in Assay Buffer.
- In a plate, load 50 µL of 8 ng/µL rmVAP-1 and start the reaction by adding 50 µL of the Substrate mixture (step 2). Include a Substrate Blank containing 50 µL of the Assay Buffer and 50 µL of the Substrate mixture.
- Read at excitation and emission wavelengths of 544 nm and 590 nm (top read), respectively in kinetic mode for 5 minutes. Note: A manual cutoff must be set at a wavelength of 570 nm.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)|
|amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using a fluorescent standard prepared by incubating 50 µM AUR, 1 unit/mL HRP, 1 mM Benzylamine, and a curve of Hydrogen Peroxide (Sigma, Catalog # H1009) in Assay Buffer. Use this oxidized AUR curve to determine the conversion factor.
- rmVAP-1: 0.4 µg
- Benzylamine: 1 mM
- HRP: 1 unit/mL
- AUR: 50 µM
Vascular adhesion protein-1 (VAP-1) is a copper amine oxidase with a topaquinone co-factor. VAP-1 is a type II integral membrane protein, but a soluble form of the enzyme is present in human serum, and its level increases in diabetes and some inflammatory liver diseases (1, 2). VAP-1 catalyzes the oxidative deamination of small primary amines such as methylamine, benzylamine, and aminoacetone in a reaction that produces an aldehyde, ammonia, and H2O2 (3). The enzyme is sensitive to inhibition by semicarbazide. VAP-1 expression is highest in the endothelium of lung, heart, and intestine, but low in tissues such as brain, spleen, kidney, and liver (4). VAP-1 vascular expression is regulated at sites of inflammation through its release from intracellular granules in which the protein is stored (5). The adhesive function of VAP-1 has been demonstrated in studies showing that the protein is important for the adherence of certain lymphocyte subtypes to inflamed endothelial tissues (6). VAP-1 mediated adhesion is involved in the process of leukocyte extravasation, an important feature of inflammatory responses. The role of VAP-1 amine oxidase activity in this process is not fully defined, but it appears to be carbohydrate-dependent (7). VAP-1 is considered to be a therapeutic target for diabetes, oxidative stress, and inflammatory diseases (8). The N‑terminal transmembrane domain of recombinant mouse VAP-1 was deleted and replaced with a signal sequence, resulting in the secretion of the soluble form of the protein.
- Kurkijärvi, R. et al. (1998) J. Immunol. 161:1549.
- Gearing, A.J.H. and W. Newman (1993) Immunol. Today 14:506.
- Lizcano, J.M. et al. (1998) Biochem. J. 331:69.
- Smith, D.J. et al. (1998) J. Exp. Med. 188:17.
- Jaakkala K. et al. (2000) Am. J. Pathol. 157:463.
- Salmi, M. and J. Jalkanen (2001) Trends Immunol. 22:211.
- Salmi, M. and J. Jalkanen (1996) J. Exp. Med. 183:569.
- Dunkel, P. et al. (2008) Curr. Med. Chem. 15:1827.
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