Recombinant Human PLA2G2A Protein, CF

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

Recombinant Human PLA2G2A Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze 1-Hexadecanoyl-2-(1-pyrene-decanoyl)-sn-glycero-3-phosphoglycerol. The specific activity is >3,750 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human PLA2G2A protein
  Asn21-Cys144, with a C-terminal 10-His tag
• Accession #: P14555
• N-terminal Sequence: Asn21
• Protein/Peptide Type: Recombinant Enzymes
• Gene: PLA2G2A
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 15 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: 17 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 and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 0.1 M NaCl, 20 mM CaCl₂, 2 mg/mL BSA, pH 8.5
  • Enzyme Buffer: 50 mM Tris, 0.1 M NaCl, 2 mg/mL BSA, pH 8.5
  • Recombinant Human Phospholipase A2 Group IIA/PLA2G2A (rhPLA2G2A) (Catalog # 5374-PL)
  • Substrate: 1-Hexadecanoyl-2-(1-Pyrenedecanoyl)-sn-Glycero-3-Phosphoglycerol Ammonium salt, 2 mM stock in DMSO
  • Black 96-well Plate
  • Fluorescent Plate Reader
  1. Thaw Substrate at 37 °C for five minutes. Mix well.
  2. Dilute rhPLA2G2A to 20 ng/mL in Enzyme Buffer.
  3. Dilute Substrate to 40 µM in Assay Buffer.
  4. Load 50 µL of the 20 ng/mL rhPLA2G2A into a black well plate, and start the reaction by adding 50 µL of 40 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 40 µM Substrate without any rhPLA2-G2A.
  5. Read at excitation and emission wavelengths of 345 nm and 395 nm (top read), respectively, in kinetic mode for 5 minutes. Mix between reads. Use the first three minutes for data analysis.
  6. 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 calibration standard 1-pyrenedecanoic acid

  Per Well:
  • rhPLA2G2A: 0.001 µg
  • Substrate: 20 µM

Notes

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

Alternate Names for Recombinant Human PLA2G2A Protein, CF

• EC 3.1.1.4
• GIIC sPLA2
• Group IIA phospholipase A2
• Mom1
• Non-pancreatic secretory phospholipase A2
• NPS-PLA2
• Phosphatidylcholine 2-acylhydrolase 2A
• phospholipase A2, group IIA (platelets, synovial fluid)
• phospholipase A2, membrane associated
• PLA2B
• PLA2BPLA2
• PLA2G2A
• PLA2L
• PLA2LPLA2S
• PLAS1
• RASF-A
• sPLA2

Background

Secretory phopholipase A₂ is an enzyme that hydrolyzes the sn-2 ester bond of phospholipids, generating non-esterified free fatty acids and lysophospholipids (1-4). 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. PLA2G2A is a calcium dependent phospholipase expressed in many cell types associated with inflammation, including platelets, neutrophils, and mast cells. It has been recognized as an acute phase reactant and its concentration increases during systemic inflammation such as sepsis, rheumatoid arthritis, and cardiovascular disease (5). It may function as an enzymatic component of the host defense mechanism. For example, human tears contain a high concentration of PLA2G2A and this is the principal bactericidal factor against Gram-positive bacteria in this fluid (1). It may play a role in cell proliferation through binding a receptor on membrane surface or extracellular matrix components such as heparan sulfate proteoglycan and integrins (6). It has been shown to have pro-atherogenic properties both in the circulation and within the arterial wall (7). Circulating levels of PLA2G2A are higher in coronary artery disease (CAD) patients and are associated with increased risk of future CAD (8).

1. Lambeau, G. and  M.H. Gelb (2008) Ann. Rev. Biochem. 77:495.
2. Webb, N.R. (2005) Curr. Opin. Lipid. 16:341.
3. Triggiani, M. et al. (2005) J. Allergy Clin. Immunol. 116:1000.
4. Murakami, M. and I. Kudo (2004) Biol. Pharm. Bull. 27:1158.
5. Crowl, R.M. et al. (1991) J. Biol. Chem. 266:2647.
6. Saegusa, J. et al. (2008) J. Biol. Chem. 283:26107.
7. de Beer, F.C. and N.R. Webb (2006) Arterioscler. Thromb. Vasc. Biol. 6:1421.
8. Wootton, P.T.E. et al. (2006) Human Mol. Genet. 15:355.

Bioinformatics

• Gene Symbol: PLA2G2A
• Uniprot:
  • P14555()