Purification and biochemical characterization of a secreted group IIA chicken intestinal phospholipase A(2)

BACKGROUND: Secretory phospholipase A2 group IIA (IIA PLA2) is a protein shown to be highly expressed in the intestine of mammals. However, no study was reported in birds. RESULTS: Chicken intestinal group IIA phospholipase A(2 )(ChPLA(2)-IIA) was obtained after an acidic treatment (pH.3.0), precipitation by ammonium sulphate, followed by sequential column chromatographies on Sephadex G-50 and mono-S ion exchanger. The enzyme was found to be a monomeric protein with a molecular mass of around 14 kDa. The purified enzyme showed a substrate preference for phosphatidylethanolamine and phosphatidylglycerol, and didn't hydrolyse phosphatidylcholine. Under optimal assay conditions, in the presence of 10 mM NaTDC and 10 mM CaCl(2, )a specific activity of 160 U.mg(-1 )for purified ChPLA(2)-IIA was measured using egg yolk as substrate. The fifteen NH2-terminal amino acid residues of ChPLA(2)-IIA were sequenced and showed a close homology with known intestinal secreted phospholipases A(2). The gene encoding the mature ChPLA(2)-IIA was cloned and sequenced. To further investigate structure-activity relationship, a 3D model of ChPLA(2)-IIA was built using the human intestinal phospholipase A(2 )structure as template. CONCLUSION: ChPLA2-IIA was purified to homogeneity using only two chromatographic colomns. Sequence analysis of the cloned cDNA indicates that the enzyme is highly basic with a pI of 9.0 and has a high degree of homology with mammalian intestinal PLA(2)-IIA.