Purification and biochemical characterization of a secreted group IIA chicken intestinal phospholipase A?.

ABSTRACT: 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? (ChPLA?-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?, a specific activity of 160 U.mg?¹ for purified ChPLA?-IIA was measured using egg yolk as substrate. The fifteen NH2-terminal amino acid residues of ChPLA?-IIA were sequenced and showed a close homology with known intestinal secreted phospholipases A?. The gene encoding the mature ChPLA?-IIA was cloned and sequenced. To further investigate structure-activity relationship, a 3D model of ChPLA?-IIA was built using the human intestinal phospholipase A? 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?-IIA.

SUBMITTER: Karray A 

PROVIDER: S-EPMC3040156 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature