tailieunhanh - Báo cáo khoa học: Biochemical and molecular characterization of purified chicken pancreatic phospholipase A2

Chicken pancreatic phospholipase A2 (ChPLA2 ) was purified from delipi-dated pancreases using ammonium sulfate and ethanol precipitation, followed by sequential column chromatography steps on MonoQ Sepha-rose and size exclusion HPLC columns. ChPLA2 was found to be a non-glycosylated monomeric protein with a molecular mass of 14 kDa and a specific activity of 400 UÆmg )1 in the presence of 1 mmsodium taur-odeoxycholate and 4 mmCaCl2with phosphatidylcholine as substrate. | ỊFEBS Journal Biochemical and molecular characterization of purified chicken pancreatic phospholipase A2 Aida Karray Fakher Frikha Abir Ben Bacha Yassine Ben Ali Youssef Gargouri and Sofiane Bezzine Laboratoire de Biochimie et de Genie Enzymatique des Lipases ENIS Sfax Tunisia Keywords bile salts chicken modeling phospholipase A2 purification Correspondence S. Bezzine Laboratoire de Biochimie et de Genie Enzymatique des Lipases ENIS Route de Soukra 3038 Sfax Tunisia Fax 216 74675055 Tel 216 74675055 E-mail sofiane_bezzine@ Database The nucleotide sequence of ChPLA2 determined in this study has been deposited in the GenBank database under the accession number EU617018 Chicken pancreatic phospholipase A2 ChPLA2 was purified from delipi-dated pancreases using ammonium sulfate and ethanol precipitation followed by sequential column chromatography steps on MonoQ Sepharose and size exclusion HPLC columns. ChPLA2 was found to be a nonglycosylated monomeric protein with a molecular mass of 14 kDa and a specific activity of 400 U-mg-1 in the presence of 1 mM sodium taur-odeoxycholate and 4 mM CaCl2 with phosphatidylcholine as substrate. The N-terminal sequence of the first 15 amino acids of ChPLA2 was determined and showed a high degree of homology with known mammal pancreatic phospholipases A2. The gene encoding the mature ChPLA2 was cloned and sequenced. The deduced amino acid sequence of the mature ChPLA2 confirmed the high level of identity with mammal pancreatic PLA2. To investigate the structure-activity relationships a 3D model of group IB ChPLA2 was built using the porcine pancreatic phospholipase A2 structure as template. Received 13 March 2009 revised 7 June 2009 accepted 17 June 2009 doi Introduction Phospholipases A2 PLA2s EC hydrolyze stereospecifically the 2-acyl ester bond of 1 2-diacyl-sn-3-phosphoglycerides generating fatty acids and lysophospholipids. Several PLA2s have been identified on the basis of their