• 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.  

• Activity

  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. See Activity Assay Protocol on .

• Source

  E. coli-derived Met33-Asn441 & Leu45-Asn441, both with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met33 & Leu45    
   

• Predicted Molecular Mass

  46 kDa

• SDS-PAGE

  42 - 45 kDa, reducing conditions

Assay Procedure

Materials

• 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:

     *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

Background: PLA2G7/PAF-AH/Lp-PLA2

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.

• References:

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.

• Long Name:

  Phospholipase A2 Group VII

• Entrez Gene IDs:

  7941 (Human); 27226 (Mouse); 301265 (Rat)

• Alternate Names:

  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