Recombinant Human PLA2G7/PAF-AH/Lp-PLA2 Protein, CF

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

Recombinant Human PLA2G7/PAF-AH/Lp-PLA2 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a colorimetric peptide substrate, 1-O-hexadecyl-2-deoxy-2-thio-S-acetyl-sn-glyceryl-3-phosphorylcholine (2-Thio-PAF), in the presence of 5,5’Dithio-bis(2-nitrobenzoic acid) (DTNB). Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468. Stewart, A.G. et al. (1991) J. Lipid Mediat 4:299. Aarsman, A.J. et al. (1991) Eur. J. Biochem 200:187. The specific activity is >9,000 pmol/min/ug, as measured under the described condition.
• Source: E. coli-derived human PLA2G7/PAF-AH/Lp-PLA2 protein
  Met33-Asn441 & Leu45-Asn441, both with a C-terminal 6-His tag
• Accession #: AAH38452
• N-terminal Sequence: Met33 & Leu45
• Protein/Peptide Type: Recombinant Enzymes
• Gene: PLA2G7

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 46 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: 42 - 45 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 Sodium Acetate, NaCl and Glycerol.
• Assay Procedure:
  • Assay Buffer: 50 mM MES, 150 mM NaCl, 0.1 mg/mL BSA, pH 6.5
  • Recombinant Human PLA2G7/PAF‑AH/Lp‑PLA2 (rhPLA2G7) (Catalog # 5106-PL)
  • Substrate: 2-Thio-PAF (Cayman Chemical, Catalog # 60945), 20 mM stock in DMSO
  • 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130), 10 mM stock in DMSO
  • 96-well clear plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhPLA2G7 to 0.1 ng/µL in Assay Buffer.
  2. Dilute Substrate to 200 μM containing 200 μM DTNB in Assay Buffer.
  3. Load 50 µL of 0.1 ng/µL rhPLA2G7 into a plate, and start the reaction by adding 50 µL of Substrate/DTNB. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate/DTNB.
  4. Read absorbance at 405 nm 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 13260 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rhPLA2G7: 0.005 µg
  • Substrate: 100 µM
  • DTNB: 100 µM

Notes

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

Alternate Names for Recombinant Human PLA2G7/PAF-AH/Lp-PLA2 Protein, CF

• 2-acetyl-1-alkylglycerophosphocholine esterase
• EC 3.1.1
• EC 3.1.1.47,1-alkyl-2-acetylglycerophosphocholine esterase
• Group-VIIA phospholipase A2
• gVIIA-PLA2
• LDL-associated phospholipase A2
• LDL-PLA(2)
• LDL-PLA2
• lipoprotein-associated phospholipase A2
• LpPLA2
• Lp-PLA2
• PAF acetylhydrolase
• PAF-AH
• PAFAHPAF 2-acylhydrolase
• phospholipase A2, group VII (platelet-activating factor acetylhydrolase
• PLA2G7
• plasma)
• platelet-activating factor acetylhydrolase

Background

Secretory phopholipase A₂ is an enzyme that hydrolyses the Sn-2 ester bond of phospholipids, generating free fatty acids and lysophospholipids (1‑3). Most secretory PLA₂s are stored in cytoplasmic granules and are released in the extracellular environment on appropriate cell activation. Thus, they are present at higher concentration in the plasma and biologic fluids of patients with systemic inflammatory, autoimmune, or allergic disease, such as acute pancreatitis, rheumatoid arthritis, bronchial asthma, and allergic rhinitis. Also known as Lp-PLA2, PLA2G-VII is a plasma enzyme bound to lipoproteins: 80% bound to LDL, 15%-20% to HDL, and the remainder to VLDL (4-6). It is produced in major by mature macrophages and activated platelets. In contrast to other classical sPLA₂s, PLA2G‑VII has poor specificity toward Sn-2 long chain fatty acids, unless heavily oxidized, and undergoes the catalysis of its substrates in the aqueous phase rather than at the interfacial surface of lipids. Thus, it has high specificity for water-soluble phospholipids in plasma including oxidatively‑modified phospholipids and platelet-activating factor (PAF). Because of the latter activity, it is also known as PAF acetylhydrolase (PAF-AH). Lack of human PLA2G‑VII is related to a higher risk for stroke and heart disease.

1. Webb, N. R. (2005) Cur. Opin. Lipid. 16:341.
2. Triggiani, M. et al. (2005) J. Allergy Clin. Immunol. 116:1000.
3. Murakami, M. and Kudo, I. (2004) Biol. Pharm. Bull. 27:1158.
4. Caslake, M. J. and Packard C. J. (2005) Nat. Clin. Prac. Cardiovasc. Med. 2:529.
5. Karabina, S.-A., and Ninio, E. (2006) Biochim. Biophys. Acta, 1761:1351.
6. Karasawa, K. (2006) Biochim. Biophys. Acta, 1761:1359.

Bioinformatics

• Gene Symbol: PLA2G7
• Uniprot:
  • AAH38452()